FROM  THE  LIBRARY  OF 
WILLIAM  A.SETCHELL,i864-i943 

PROFESSOR  OF  BOTANY 


GY  LIBRARY 


. 

Sexual  Reproduction  and  the  Organization  of 
the  Nucleus  in  Certain  Mildews. 


I     ' 


BY 


R.  A.  HARPER 


WASHINGTON,  D.  C. : 

Published  by  the  Carnegie  Institution  of  Washington 
October,  1905. 


W 


Sexual  Reproduction  and  the  Organization  of 
the  Nucleus  in  Certain  Mildews. 


BY 


R.  A.  HARPER 


WASHINGTON,  D.  C.: 

Published  by  the  Carnegie  Institution  of  Washington 
September,  1905. 


Nit 

•dftl. 
Lib, 


CARNEGIE  INSTITUTION  OF  WASHINGTON 
PUBLICATION  No.  37 


BIOLOGY  LIBRARY 


FROM  THE  PRESS  OF 

THE  HENRY  E.  WILKENS  PRINTING  CO. 

WASHINGTON,  D.  C. 


CONTENTS. 


Page. 

PREFACE ....  3 

INTRODUCTION 5 

DEVELOPMENT  OF  THE  PERITHECIUM — 

The  mycelium  and  the  origin  of  oogonium  and  antheridium 8 

Fertilization 10 

Degeneration  of  antheridial  wall  and  cytoplasm 12 

Development  of  the  ascogonium  and  origin  of  the  asci  as  binucleated  cells. . .  15 

Secondary  mycelium,  appendages,  and  penicillate  cells 21 

Function  of  appendages  and  period  of  spore  formation 24 

MORPHOLOGY  OF  THE  ASCOCARP — 

Review  of  recent  work  on  the  development  of  various  Ascomycetes 29 

Dangeard  's  conception  of  the  gametophore 28 

Conclusions  as  to  morphology  of  ascocarp  and  asci 29 

SPECIAL  NUCLEAR  PHENOMENA — 

Relative  size  of  nuclei  and  cells  in  the  ascocarp 30 

Structure  of  nuclei  and  relations  of  central  body  and  chromatin  in  gametes 

and  ascogenous  cells 31 

Nuclear  fusion  in  ascus 36 

Synapsis,  spirem,  number  of  chromatin  filaments 39 

Spindle  formation,  equatorial  plate,  and  anaphases 43 

Formation  of  the  daughter  nuclei 46 

Second  and  third  divisions 47 

Development  of  ascospores  by  free  cell-formation 48 

Summary  of  relation  of  central  body  and  chromosomes  in  the  development 

of  the  ascocarp 52 

THEORETICAL  DISCUSSION  : 

THE  CENTRAL  BODY  IN  PHYLLACTINIA — 

Polar  organization  of  the  nucleus 53 

Central  body  a  permanent  structure 54 

PERMANENCE  OF  THE  CHROMOSOMES — 

The  organization  of  the  chromatin  in  resting  nuclei  and  segregation  of  the 

chromosomes 56 

Individuality  of  chromosomes 57 

Review  of  data  as  to  connection  of  chromosomes  and  centrosome 59 

THE    NUCLEAR    FUSION    IN    THE    ASCUS 

Comparison  with  fusion  in  oogonium  and  in  aecidium  of  rusts 61 

Doctrine  of  the  nucleo-cytoplasmic  relation 64 

Relation  of  assimilation  and  growth  to  cell  and  nuclear  division 67 

Conclusions  as  to  origin  and  nature  of  fusion  in  ascus 71 

Effects  of  development  of  conjugate  nuclear  division 72 

i 


2  CONTENTS. 

Page. 
RELATIONS  OF  ASCOMYCETES  AND  BASIDIOMYCETES — 

Evidence  from  binucleated  cells  for  relations  of  rusts  and  Basidiomycetes. . .  76 
Resemblances  in  Ascomycetes  and  Basidiomycetes  due  to  parallelism  in  func- 
tions    77 

ALTERNATION  OF  GENERATIONS  IN  HIGHER  FUNGI — 

Evidence  from  reduction  phenomena  and  binucleated  cells 79 

Correlation  of  triple  division  in  ascus  with  two  preceding  nuclear  fusions  ...  81 

Relations  of  ascus,  teleutospore,  basidium,  and  tetrasporange 85 

Summary  as  to  sexual  reproduction  and  alternation  of  generations  in  higher 

fungi 86 

INDEX  OF  LITERATURE 88 

EXPLANATION  OF  FIGURES  IN  PLATES 92 


PREFACE. 

The  organization  of  the  resting  nucleus  and  its  relation  to  the  proc- 
esses of  nuclear  fusion  and  division  are  the  main  problems  with  which 
I  have  been  concerned  in  continuing  my  studies  on  the  mildews.  The 
doctrine  is  commonly  accepted  that  the  chromosomes  are  in  a  special 
sense  the  physical  basis  of  heredity,  but  their  relation,  especially  in  the 
resting  nucleus,  to  the  mechanism  of  cell  division  and  to  the  centrosome 
is  still  undetermined.  The  evidence  for  the  so-called  "  individuality  " 
of  the  chromosomes  has  been  developed  almost  entirely  from  a  study  of 
their  appearance  in  mitosis.  In  Phyllactinia  I  have  been  able  to  show 
that  the  material  of  each  chromosome  is  in  permanent  connection  with 
the  central  body  throughout  the  stages  of  nuclear  fusion  and  the  resting 
condition,  as  well  as  in  mitosis,  thus  affording  an  explanation  of  the 
means  by  which  the  permanence  of  the  chromosomes  is  secured  and 
throwing  further  light  on  the  mechanics  of  nuclear  division.  The 
nucleus  is  thus  shown  to  have  a  permanent  polar  organization ;  and  the 
central  body  is  a  permanent  structure  in  the  cell  which  determines  the 
point  of  special  connection  between  the  nuclear  contents  and  the  cyto- 
plasm in  the  mildews.  The  regularly  recurring  triple  nuclear  division 
in  the  ascus  has  been  the  most  serious  objection  to  the  acceptance  of  the 
frequently  proposed  view  that  the  ascocarp  is  a  sporophyte  and  the 
ascus  a  spore  mother  cell.  But  with  the  facts  of  the  nuclear  history  in 
Phyllactinia  here  brought  out,  showing  that  each  chromosome  is  a  per- 
manent unit  through  the  processes  of  nuclear  fusion  as  well  as  division, 
and  that  synapsis  and  a  numerical  reduction  of  the  chromosome  number 
occur  in  the  ascus,  it  becomes  plain  that  the  triple  division  in  the  ascus  is 
a  natural  correlative  of  the  occurrence  of  two  nuclear  fusions  in  the  de- 
velopment of  the  ascocarp.  The  evidence  thus  becomes  very  strong  that 
we  have  a  true  alternation  of  generation  in  the  Ascomycetes.  In  the 
light  of  the  principle  of  the  "  nucleo-cytoplasmic  relation  "  it  is  also 
evident  that  the  size,  method  of  development,  and  functions  of  the  ascus 
all  indicate  that  the  nuclear  fusion  which  occurs  in  it  is  a  correlative  of 
the  general  vegetative  and  growth  processes  involved  in  maintaining 
the  nucleo-cytoplasmic  equilibrium  in  a  cell  of  such  relatively  gigantic 
size  as  the  ascus. 

The  presence  of  typically  differentiated  and  functional  sexual  cells  in 
Phyllactinia,  together  with  the  abundant  evidence  which  has  recently 


4  PREFACE. 

accumulated,  and  is  here  summarized,  as  to  their  presence  in  other  types, 
must  be  regarded  as  establishing  the  fact  that  the  ascocarp  originates  in 
a  sexual  apparatus.  Objections  which  are  still  urged  by  the  opponents 
of  De  Bary's  views  are  rather  in  the  nature  of  complaints  and  denials 
than  in  the  presentation  of  new  facts. 

A  most  fertile  source  of  confusion  in  the  discussion  of  the  relation- 
ships* of  fruit  forms  in  the  fungi  has  been  the  failure  to  make  all  com- 
parisons from  the  standpoint  of  a  strictly  phylogenetic  morphology. 
Whatever  excuse  there  may  be  for  introducing  physiological  concep- 
tions into  the  formal  classification  of  tissues  in  the  higher  plants,  there 
can  be  no  question  that  great  confusion  has  arisen  in  the  morphology 
of  the  fungi  and  algae  from  allowing  considerations  of  functional 
equivalence  or  difference  to  mingle  with  and  modify  the  conceptions 
of  what  should  be  a  strictly  phylogenetic  morphology.  It  may  be  a 
hopeless  task — it  certainly  is  not  at  present  a  very  important  or  stim- 
ulating one — to  attempt  to  determine  with  exactness  lines  of  phylogenetic 
descent  among  such  plastic  forms  as  the  algae  and  fungi ;  but  no  other 
or  lesser  aim  than  this  should  be  allowed  to  masquerade  under  the  guise 
of  morphology.  Physiological  data  as  to  the  functions  and  mechanics 
of  cells  and  organisms  are  of  far  greater  biological  significance  than 
those  of  phylogenetic  morphology,  but  this  is  no  reason  for  mixing  the 
two  or  allowing  such  attempts  as  we  may  make  at  the  arrangement  of 
plant  forms  in  evolutionary  series  to  be  vitiated  by  the  introduction  of 
purely  functional  criteria  into  the  determination  of  our  morphological 
classifications.  The  question,  for  example,  whether  a  given  organ  is 
functional  in  its  more  primitive  or  in  some  modified  fashion  can  have  no 
bearing  upon  the  determination  of  its  morphological  nature  or  rela- 
tionships. 

Comparisons  must  frequently  be  made  and  hypothetical  morpho- 
logical categories  established  on  data  which  are  incomplete  and  can, 
perhaps,  never  be  made  complete,  but  a  lack  of  data  as  to  phylogenetic 
relationship  can  never  be  compensated  for  by  evidence  of  functional 
equivalence  or  non-equivalence.  The  modern  fields  of  causal  and 
experimental  morphology,  developmental  mechanics,  etc.,  belong  with 
the  physiological  rather  than  the  morphological  disciplines,  and  their 
results  have  the  same  bearing  upon  the  classifications  of  phylogenetic 
morphology  as  have  other  physiological  data.  If  there  can  be  agree- 
ment that  the  various  developmental  stages  and  fruit  forms  of  the  fungi 
and  algae  shall  be  classified  and  named  in  accordance  with  what  can  be 
determined  as  to  their  phylogeny  a  number  of  disputed  questions  will 
disappear. 


SEXUAL  REPRODUCTION  AND  THE  ORGANIZATION  OF 

THE  NUCLEUS  IN  CERTAIN  MILDEWS. 


BY  R.   A.   HARPER. 


INTRODUCTION. 

It  is  one  of  the  best-established  facts  regarding  mitosis  that  from 
certain  late  prophase  stages  until  the  formation  of  the  diaster  a  specially 
evident  connection  between  the  chromosomes  and  the  spindle  poles  is 
maintained  by  means  of  conspicuous  fibrillar  structures.  The  existence 
of  these  apparent  fibrillse  is  generally  admitted  even  by  those  who  accept 
a  so-called  dynamic  theory  of  the  central  body  as  opposed  to  the  concep- 
tion of  contractile  spindle  and  polar  fibers.  It  is  evident,  also,  that  this 
connection  influences,  if  not  directly  determines,  the  motions  of  the 
chromosomes  and  their  separation  into  two  groups  to  form  the  daughter 
nuclei.  This  is  equally  true  for  the  cells  of  animals  and  plants  and  for 
cases  of  multipolar  as  well  as  bipolar  spindles. 

Rabl  was  the  first  to  point  out  that  in  the  spirem,  and  even  in  the 
earlier  resting-stages,  some  influence  is  exerted  on  the  nuclear  contents 
which  gives  a  definite  polar  arrangement  to  the  chromatic  elements. 
This  centering  of  the  spirem  loops  on  a  polar  field  has  come  to  be  com- 
monly known  as  Rabl's  (78)  figure.  The  same  arrangement  of  the 
spirem  was  found  by  Strasburger  (88)  in  both  the  dispirem  and  the 
prophases  of  dividing  plant-cells,  and  its  regular  occurrence  has  since 
been  described  and  figured  by  numerous  authors  and  for  widely  sepa- 
rated organisms. 

The  discovery  of  the  so-called  attraction  sphere  and  its  relation  to 
the  karyokinetic  figure  by  Van  Beneden  (6,7)  gave  a  further  impulse  to 
the  doctrine  of  polarity,  both  in  the  cell  and  nucleus,  and  in  a  further 
brief  paper  (79)  Rabl  accepted  Van  Beneden's  conception  of  an  attrac- 
tion sphere  and  gave  a  fuller  statement  of  the  doctrine  that,  not  only 
during  the  process  of  division  as  shown  by  Van  Beneden,  but  in  the 
so-called  resting  stages,  the  whole  structure  of  the  cell  and  nucleus  is 

NOTE.— Numbers  inclosed  in  parentheses  refer  to  the  numbers  in  the  litera- 
ture list  at  the  end  of  this  paper. 

5 


6  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

centered  on  the  polar  body  in  the  polar  field  of  the  cytoplasm.  He 
described  the  nucleus  as  showing  thus  a  polar  and  an  antipolar  region 
and  its  whole  structure,  including  both  chromatic  and  achromatic  ele- 
ments, as  permanently  centered  on  a  definite  pole.  Rabl  further  con- 
ceives the  achromatic  fibers  of  the  resting  condition  as  forming  the 
spindle  figure  in  karyokinesis,  and  thus  the  basis  is  given  for  a  mechan- 
ical conception  of  division  on  the  assumption  of  the  contractility  of  the 
fibrillar  elements  of  the  nucleus  and  cytoplasm. 

Rabl's  conception  of  the  polar  organization  of  the  nucleus  and  of 
the  mechanics  of  nuclear  division  was  adopted  by  Flemming  and  is 
given  its  fullest  expression  in  his  discussion  of  the  mechanics  of  cell 
division  in  the  tissue  cells  of  the  salamander  (25,  pp.  715-749)- 

Boveri  (10),  on  the  ground  of  his  observations  on  the  eggs  of 
Ascaris,  opposed  the  doctrine  of  a  persistent  connection  between  tne 
chromatic  elements  and  the  centrosome,  and  maintained  that  a  new  con- 
nection between  chromosomes  and  poles  is  established  in  the  prophases 
of  each  succeeding  cell  division.  With  the  development  of  the  doctrine 
of  the  individuality  of  the  chromosomes  this  question  has  gained  still 
more  fundamental  importance,  and  in  this  connection  I  shall  discuss, 
further  on,  the  observations  of  some  later  investigators. 

My  earlier  work  (38)  on  the  dividing  nuclei  in  the  ascus  led  me 
to  suspect  that  a  permanent  connection  of  chromatin  elements  and  cen- 
tral body  exists  in  the  mildews,  and  I  have  described  and  figured  such 
a  condition  in  certain  resting  stages  of  the  nuclei.  In  extending  my 
studies  to  the  genus  Phyllactinia  I  have  found  a  more  favorable  form 
with  somewhat  larger  nuclei  and  have  been  able  to  trace  a  continuous 
connection  of  centers  and  chromatin  in  both  resting,  fusing,  and  dividing 
nuclei. 

Since  various  authors  have  expressed  themselves  as  still  denying 
or  as  doubtful  as  to  the  existence  of  differentiated  sexual  cells  in  the 
Ascomycetes  and  as  to  the  consequent  morphological  relations  of  the 
ascocarp,  I  shall  precede  my  special  description  of  the  nuclear  structures 
and  processes  with  an  account  of  the  development  of  the  sexual  organs 
and  the  perithecium.  I  am  convinced  that  on  these  much-vexed  ques- 
tions of  the  alternation  of  generations,  relations  of  Ascomycetes,  Basidi- 
omycetes,  red  algae,  etc.,  we  shall  only  arrive  at  certainty  by  the  most 
painstaking  study  of  the  nuclear  structures  and  processes  and  especially 
the  chromosome  number  at  all  stages  of  development,  and  I  have 
returned  to  the  mildews  for  a  further  contribution  to  this  subject  because 
in  Phyllactinia  I  have  found  the  most  favorable  ascomycete  for  the 
study  of  the  nuclei  which  I  have  yet  encountered.  Phyllactinia  is 


INTRODUCTION.  7 

undoubtedly  the  most  highly  specialized  of  the  mildews  and  represents 
the  culmination  of  the  developmental  tendencies  found  in  the  group. 
It  is  one  of  our  most  widely  distributed  mildews  and  has  been  repeat- 
edly the  object  of  investigation  from  the  standpoint  of  its  general  struc- 
ture and  habits  of  life.  Recently  Salmon  (82,  p.  224)  has  investigated 
very  fully  the  published  species  of  the  genus,  and  has  concluded  that 
we  have  but  one  polymorphic  and  cosmopolitan  type.  He  adopts  the 
name  Phyllactinia  corylea  (Pers)  Karst. 

Salmon  (83,  pp.  201-205)  has  also  made  very  extensive  and  inter- 
esting comparative  studies  of  the  form  of  the  peculiar  penicillate  cells, 
and  concludes  that  the  most  extreme  types  of  these  cells  are  connected 
by  a  series  presenting  all  possible  intermediate  variations  in  form.  Palla 
(76)  and  Smith  (86)  discovered  the  interesting  fact  that  Phyllactinia, 
unlike  other  mildews,  is  not  strictly  epiphytic,  but  sends  branches 
through  the  stomata  into  the  intercellular  spaces  of  the  mesophyl,  pro- 
ducing haustoria  ultimately  in  the  cells  at  the  base  of  the  palisade 
parenchyma,  and  especially  where  nutrition  is  most  abundant.  Neger 
(68)  has  also  corrected  the  erroneous  view  that  the  conidia  are  borne 
singly,  instead  of  in  chains  as  in  other  mildews,  and  has  given  very 
interesting  data  as  to  the  operation  of  the  spine-like  appendages,  which, 
by  their  hygroscopic  motions,  loosen  and  lift  up  the  perithecia  from  the 
leaf  of  the  host  when  they  are  ripe. 

Phyllactinia  occurs  on  a  considerable  series  of  host  plants.  I  have 
sectioned  and  studied  especially  material  from  the  white  ash  (Fraxinus 
americana),  the  hazel  (Corylus  americana},  the  bittersweet  (Celastrus 
scandens),  and  the  yellow  birch  (Betula  lutea).  The  material  was 
fixed,  immediately  upon  being  gathered  in  the  field,  and  its  subsequent 
treatment  was  essentially  the  same  as  I  have  previously  described. 


8  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 


DEVELOPMENT   OF  THE    PERITHECIUM. 

The  sexual  apparatus  in  Phyllactinia  arises  in  general  as  I  have 
earlier  described  for  Sphaerotheca  (36)  and  Erysiphe  (37).  As  a  rule 
the  ascocarps  are  more  sparsely  scattered  than  in  either  of  the  above 
genera,  and  the  labor  of  bringing  together  a  series  of  developmental 
stages  is  proportionally  increased.  As  has  been  many  times  noted,  the 
perithecia  of  Phyllactinia  are  regularly  hypophyllous,  a  fact  which  has 
its  explanation  in  the  existence,  as  above  noted,  of  interior  mycelial 
branches  which  gain  access  to  the  mesophyl  through  the  stomata  of  the 
host.  This  habit  brings  with  it  further  a  relatively  slight  development  of 
the  superficial  mycelium,  and  hence  the  infected  spots  are,  as  compared 
with  those  of  most  mildews,  extremely  inconspicuous  until  the  young 
fruit  bodies  have  grown  large  enough  to  be  visible  to  the  naked  eye  and 
have  taken  on  the  characteristic  yellowish  color  of  young,  half-devel- 
oped mildew  perithecia.  At  stages  when  fertilization  is  going  on,  the 
infected  spot  on  the  ash  or  bittersweet  leaf  is  faintly  visible  as  a  slightly 
whitened  region  scarcely  noticeable  unless  the  leaf  is  held  so  that  the 
light  is  reflected  from  its  surface  at  a  particular  angle.  In  my  experi- 
ence conidiophores  are  almost  or  entirely  lacking,  even  in  the  earliest 
stages,  on  the  mycelia  which  produce  the  most  abundant  perithecia.  It 
should  be  said,  however,  that  I  have  uniformly  obtained  my  best  material 
of  this  fungus  in  the  autumn,  and  in  regions  where  the  fungus  develops 
richly  earlier  in  the  season  a  greater  abundance  and  prevalence  of  the 
conidiophores  may  be  expected. 

It  is,  of  course,  well  known  that  not  all  the  perithecia  on  an  infected 
spot  develop  simultaneously.  In  Sphaerotheca,  as  I  have  already  de- 
scribed (36),  there  is  a  considerable  peripheral  growth  of  the  mycelium, 
and  the  perithecia  may  be  half-grown  in  the  center  of  a  spot  while  an 
abundance  of  the  younger  stages  is  still  to  be  found  on  the  periphery. 
In  Phyllactinia,  however,  this  is  at  least  not  so  notably  the  case;  the 
mycelium  on  an  infected  area  seems  to  get  almost  its  full  development 
before  the  perithecia  begin  to  appear,  and  they  are  then  formed  scatter- 
ingly,  more  or  less,  over  the  whole  spot ;  the  later- formed  fruits  are  thus 
mingled  with  the  earlier.  The  most  abundant  fertilization  stages  in 
my  experience  are  to  be  found  on  spots  in  which  the  earliest-formed 
perithecia  appear  as  mere  white  specks  under  the  magnifier  and  none 
are  yet  far  enough  developed  to  have  begun  to  turn  yellow. 


DEVELOPMENT    OF    THE    PERITHECIUM.  9 

The  sexual  apparatus  is  formed,  as  in  other  mildews,  where  two 
hyphae  cross  or  lie  close  beside  each  other,  and  we  have  thus  the  oogonia 
and  antheridia  arising  as  lateral  branches  from  separate  hyphae,  though 
it  seems  fairly  clear,  from  the  circular  shape  of  the  infected  spots  in 
most  cases  in  Phyllactinia,  that  both  these  hyphae  belong  to  a  single 
mycelium,  which  in  all  probability  is  the  product  of  the  growth  of  a 
single  spore. 

The  oogonium  and  the  antheridial  branch  seem  to  arise  simulta- 
neously, and  they  become  closely  applied  to  each  other  and  begin  to 
become  slightly  spirally  twisted  at  once.  The  oogonium  is  thicker  and 
heavier  from  the  start  and  grows  in  length  more  rapidly  also,  so  that  it 
bends  around  the  antheridial  branch  while  the  latter  remains  straighter. 
The  oogonium  may  make  one  almost  complete  turn  about  the  antheridial 
branch,  which  tends  to  stand  rather  vertically  to  the  surface  of  the  leaf 
(figs.  2,  3,  10).  The  oogonium  is  much  thickened  in  the  middle  and 
may  taper  considerably  toward  both  its  base  and  apex  (fig.  i).  It  is 
densely  filled  with  protoplasm  and  contains  regularly  a  single  nucleus 
(figs,  i,  3-7) .  It  is  cut  off  from  the  hypha,  on  which  it  arose  as  a  lateral 
branch,  by  a  cross-wall  which  is  put  in  a  short  distance  from  its  point 
of  origin  on  the  mother  hypha.  The  mycelial  cell  from  which  the  oogo- 
nium arises  is  generally  of  average  length,  and  the  oogonial  branch  may 
arise  at  either  end  or  in  the  middle  of  such  a  cell  (fig.  2) . 

There  is  no  evidence  of  any  special  differentiation  in  the  cell  from 
which  an  oogonium  arises,  and  the  first  division  of  the  nucleus  of  such 
a  cell  furnishes  one  daughter  nucleus  for  the  gamete  and  the  other  for 
the  cell  of  the  mother  hypha.  The  antheridial  branch  arises  in  the  same 
fashion,  but  is  slenderer  at  the  start,  and  there  is  apparently  no  pushing 
up  of  the  antheridial  branch  by  the  side  of  the  already  developed  oogo- 
nium, as  I  have  described  for  Sphaerotheca.  Indeed,  it  seems  as  if  the 
two  branches  were  firmly  attached  at  a  very  early  stage  and  that  this 
condition,  combined  with  the  more  rapid  elongation  of  the  oogonium, 
leads  to  the  bending  of  the  latter  around  the  antheridial  branch.  This 
stretching  of  the  oogonium  also  bends  the  tip  of  the  antheridial  branch 
to  one  side  and  twists  it  slightly,  so  that  it  comes  to  lie  on  the  upper  side 
01  the  end  of  the  oogonium  (figs.  3,  4,  5,  8,  10),  not  forming  a  cap  on 
its  apex,  as  in  Sphaerotheca. 

It  is  to  be  noted  that  the  unequal  tendency  to  spiral  twisting  in  the 
sexual  branches,  together  with  their  unequal  size,  produces  a  structure 
whose  appearance  varies  considerably,  according  to  the  side  and  the 
angle  from  which  it  is  viewed,  as  is  well  seen  by  comparing  figs,  i  to  n, 
which  serve  to  show  the  various  possible  aspects  as  well  as  the  different 


IO  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

stages  of  development  of  the  sexual  apparatus.  It  is  not  easy  to  bring 
out  by  shading  the  various  bendings  of  the  cells,  as  they  can  be  fol- 
lowed by  focusing  up  and  down  through  the  thickness  of  the  section, 
and  at  the  same  time  to  show  the  contents  of  every  part  of  each  cell  as  it 
appears  in  median  optical  section.  For  the  most  part  I  have  contented 
myself  here,  as  in  my  earlier  figures  of  Erysiphe,  with  fulfilling  the 
second  and,  of  course,  the  more  important  of  the  two  requirements. 
Hence  the  figures,  with  the  partial  exception  of  2  and  3,  are  to  be 
regarded  as  representing  projections  of  the  outlines  of  the  cells  as  they 
are  followed  by  focusing  up  and  down  through  the  section,  with  the 
content  of  each  cell  as  shown  in  median  optical  section.  As  a  further 
result,  the  relative  length  of  the  cells  in  each  section  and  in  successive 
figures  is  not  in  every  case  correctly  indicated.  In  a  majority  of  cases 
the  long  axis  of  the  oogonium  tends  to  be  parallel  to  the  surface  of  the 
leaf  or  to  rise  slightly  at  its  outer  end,  as  shown  in  figs.  2,  3,  4,  10,  while, 
as  noted  above,  the  antheridial  branch  is  more  nearly  vertical  to  the 
leaf  surface.  Many  exceptions  to  these  relations  are,  however,  to  be 
found,  as  shown  in  the  other  figures  of  these  stages. 

Fig.  i  shows  a  stage  when  the  antheridial  branch  is  not  yet  cut  off 
from  the  hyphal  cell  from  which  it  arose,  and  it  contains  a  single  nucleus. 
This  nucleus  divides,  and  a  cell-wall  is  formed  between  the  daughter 
nuclei,  so  that  one  of  them  becomes  the  nucleus  of  the  antheridial 
branch  and  the  other  remains  in  the  hyphal  cell  below  (fig.  3).  This 
cross-wall  is  put  in  quite  constantly  at  the  narrowed  region  where  the 
antheridial  branch  is  partially  encircled  by  the  oogonium  (figs.  3,  10). 
The  nucleus  of  the  antheridial  branch  now  divides,  and  one  of  the 
daughter  nuclei  migrates  into  the  tip  of  the  branch  (fig.  4).  The  tip  is 
cut  off  by  a  cross-wall  and  becomes  the  antheridial  cell  (figs.  5,  6,  7). 

At  this  stage  no  trace  of  the  enveloping  perithecial  hyphse  can  be 
found.  The  whole  apparatus  consists  only  of  the  two  gametophores 
and  the  gametes  which  have  developed  almost  simultaneously  and  are 
thus  sharply  distinguished  in  the  time  of  their  development  from  the 
protective  enveloping  hyphal  branches  which  come  later.  In  Phyllac- 
tinia,  as  in  Sphaerotheca  and  Erysiphe,  the  claim  that  the  antheridial 
branch  is  to  be  interpreted  as  merely  the  first  of  the  protective  hyphse 
is  negatived  by  its  appearing  almost  simultaneously  with  the  oogonium, 
while  the  protective  hyphse  arise  much  later  and  not  singly,  but  in  series 
around  the  base  of  the  sexual  branches. 

Fertilization  occurs,  as  in  Sphaerotheca  and  Erysiphe,  by  the  break- 
ing down  of  the  walls  separating  the  protoplasts  of  the  oogonium  and 
antheridium  and  the  fusion  of  the  two  protoplasts,  and  ultimately  their 


DEVELOPMENT    OF    THE    PERITHECIUM.  II 

nuclei.  Figs.  5,  6,  and  7  show  successive  stages  after  the  gametes  have 
been  differentiated  and  just  before  fertilization  occurs.  The  oogonium 
especially  grows  in  size,  and  both  the  egg  and  male  nuclei  also  increase 
in  size.  At  the  time  of  fertilization  the  male  nucleus  is  smaller  than 
the  egg-nucleus,  but  hardly,  perhaps,  to  the  same  degree  that  the  anther- 
idial  cell  is  smaller  than  the  oogonium.  The  cytoplasm  of  both  gametes 
is  quite  free  from  reserve  food  granules  or  other  stainable  inclusions  at 
this  stage.  Some  small  bodies,  staining  red  with  the  triple  stain,  and 
a  good  many  small  spherical  vacuoles  are  present  in  the  oogonium,  as 
shown  in  the  figures,  but  for  the  most  part  the  cytoplasm  is  a  fine- 
meshed,  spongy  mass,  which  appears  pale-bluish  or  gray  with  the  triple 
stain.  The  nuclei  are  very  sharply  and  characteristically  differentiated 
at  this  stage,  and  their  structure  will  be  described  in  detail  later.  The 
antheridium  is  closely  pressed  upon  the  oogonium  a  little  to  one  side  of 
and  generally  above  the  apex  of  the  latter.  Their  walls  in  the  region 
of  contact  are  flattened  upon  each  other  and  closely  united.  Every 
appearance  is  similar  to  those  found  in  other  cases  of  non-motile  conju- 
gating cells  among  the  algae  and  fungi. 

A  portion  of  the  walls  between  the  antheridium  and  the  oogonium 
is  now  dissolved  in  such  a  fashion  as  to  form  a  circular  conjugation- 
pore  leading  from  the  antheridium  to  the  oogonium.  The  protoplasts 
of  the  antheridial  and  oogonial  cells  are  thus  brought  into  direct  contact 
and  combine  to  form  a  continuous  protoplasmic  mass  (figs.  8,  9).  The 
nucleus  of  the  antheridium  migrates  through  the  conjugation-pore  into 
the  oogonium  and  approaches  the  egg-nucleus.  The  male  nucleus  is, 
at  this  stage  also,  somewhat  smaller  than  the  egg-nucleus.  This  inequal- 
ity in  size  is,  however,  by  no  means  so  great  as  is  found  in  the  pronuclei 
of  many  other  plants  and  animals,  in  the  early  stages  of  the  process 
of  fertilization.  Figs.  8  and  9  show  cases  in  which  the  conjugation- 
pore  is  open  and  the  male  and  female  nuclei  are  side  by  side  in  the 
oogonium.  In  fig.  9  the  male  nucleus,  as  recognized  by  its  smaller  size, 
has  moved  past  and  appears  on  the  side  of  the  egg-nucleus  farthest  from 
the  antheridium. 

It  is  evident  that  the  conjugation-pore  can  be  most  easily  found 
and  studied  in  cases  in  which  the  plane  of  contact  of  the  antheridium 
and  oogonium  cut  the  plane  of  the  section  at  right  angles,  or  nearly  so, 
as  is  the  case  in  figs.  9,  10,  and  n.  Cases  in  which  the  sexual  cells 
present  the  aspects  shown  in  figs.  6  and  7,  in  which  the  antheridium  is 
more  or  less  behind  the  tip  of  the  oogonium,  are  of  course  much  less 
favorable  for  the  discovery  and  study  of  the  actual  fusion  of  the  cells. 
Still  it  is  perfectly  possible  in  well-fixed  and  clearly  stained  material  to 


12  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

demonstrate  the  conjugation-pore  from  this  aspect  also.  Fig.  8  shows 
such  a  case  in  which  the  surface  of  contact  of  the  gametes  lies  almost 
exactly  in  the  plane  of  the  section.  The  conjugation-pore  appears  here 
as  a  clean-cut  opening  in  the  floor  of  the  antheridium.  It  is  a  perfectly 
clear,  circular  area,  sharply  distinguished  from  the  bluish  material  of 
the  cell-walls  which  surround  it.  This  figure,  in  the  region  of  the 
antheridium,  represents  an  optical  section  lying  in  the  plane  of  contact 
of  the  gametes,  and  the  existence  of  an  open  pore  from  the  antheridium 
to  the  oogonium  is  unmistakable.  It  is  evident  from  the  description 
given  above  that  the  shape  and  relations  of  the  gametes  are  such  that, 
with  any  possible  orientation  of  the  host-leaf  to  the  plane  of  the  sections, 
the  aspects  shown  in  figs.  6,  7,  and  8  will  be  quite  as  common  as  those 
shown  in  figs.  9,  10,  and  n.  The  necessity  for  finding  this  latter  aspect 
of  the  gametes  in  order  to  demonstrate  the  process  of  fusion  with  the 
greatest  ease  is  doubtless  the  reason  why  some  observers  have  failed  to 
convince  themselves  that  a  union  of  the  gametes  actually  occurs.  Fig.  9 
shows  this  latter  aspect  of  the  fusing-cells.  The  plane  of  contact  of  the 
gametes  cuts  the  plane  of  the  section  almost  exactly  at  right  angles,  and 
the  conjugation-pore  appears  as  an  absolutely  unmistakable  open  pass- 
ageway leading  from  the  antheridium  to  the  oogonium;  and  the  cyto- 
plasmic  contents  of  the  gametes  are  seen  to  have  fused  to  form  a  con- 
tinuous protoplasmic  mass  through  which  the  male  nucleus  has  migrated 
into  the  oogonium.  The  two  pronuclei  lie  side  by  side  in  the  oogonium 
and  are  readily  distinguishable  by  their  difference  in  size.  In  this  par- 
ticular case  a  vacuole  lies  just  outside  the  conjugation-pore  in  the  anthe- 
ridium, indicating,  perhaps,  a  relative  increase  in  the  cell  sap  of  the 
latter  after  the  departure  of  the  male  nucleus. 

The  entire  contents  of  the  antheridial  cell  does  not  enter  the  oogo- 
nium. In  the  old  antheridium,  after  fertilization  is  complete  there  is 
considerable  material  which  degenerates  into  a  dense,  structureless  mass. 
In  fact,  it  seems  quite  likely  that  here,  as  in  many  other  cases,  only  a 
minimal  amount,  if  any,  of  the  cytoplasm  of  the  male  cell  is  actually 
taken  up  into  the  egg.  The  male  nucleus  apparently  leaves  the  most,  if 
not  all,  of  its  cytoplasm  behind  when  it  enters  the  egg.  The  cytoplasm 
of  the  antheridium  is  certainly  less  dense  and  is  more  vacuolar  for  a 
time  after  fertilization  has  taken  place.  A  few  deeply-stained  granules 
are  present  in  the  antheridium  while  the  conjugation-pore  is  still  open 
(fig.  9)  and  are  perhaps  the  first  indication  of  the  degeneration  of 
its  protoplasm.  After  the  closing  of  the  conjugation-pore  they  may 
become  more  numerous  (fig.  10),  but  the  antheridial  cytoplasm  keeps  its 
spongy  appearance  for  a  time  (fig.  n).  Soon  the  whole  of  the  cyto- 


DEVELOPMENT    OF    THE    PERITHECIUM.  13 

plasm  becomes  a  dense,  shrunken,  highly  stainable  mass,  as  will  be 
described  in  more  detail  a  little  later. 

It  is  to  be  noted  that  in  Phyllactinia  there  is  no  lack  of  granular 
material  in  the  disintegrating  cytoplasm  of  the  antheridium  to  represent 
a  degenerating  nucleus  for  one  who,  like  Dangeard,  has  failed  to  find 
the  real  process  of  fertilization.  Apparently  the  fusion  of  the  pro- 
nuclei  occurs  in  about  the  center  of  the  oogonium.  The  fusion  nucleus 
is  slightly  larger  than  either  of  the  pronuclei  (figs.  10,  n)  and  may  show 
an  increased  density  of  its  chromatin  for  a  time  (fig.  10).  Details  of 
the  fusion-process  and  the  union  of  the  different  parts  of  the  pronuclei 
in  the  fusion-nucleus,  so  far  as  I  have  been  able  to  observe  them,  are 
presented  later  in  connection  with  my  studies  of  the  fusion  of  the  nuclei 
in  the  ascus.  After  fusion  the  nucleus  lies  in  the  middle  region  of  the 
oogonial  cell  (figs.  10-12). 

The  conjugation-pore  leading  from  the  antheridium  into  the  egg 
cell  is  closed  at  once  after  the  passage  of  the  male  nucleus  (fig.  10)  and 
the  antheridial  cell  now  undergoes  some  very  interesting  and  character- 
istic degenerative  changes  which  make  it  a  most  conspicuous  object  all 
through  the  earlier  development  of  the  ascocarp,  and  which  in  them- 
selves are  conclusive  evidence  that  it  is  a  differentiated  gamete-cell 
differing  entirely  in  its  nature  from  the  cells  of  the  perithecial  envelopes. 
These  changes  consist  in  a  swelling  and  change  in  composition  of  the 
antheridial  wall  and  the  shrinkage  of  the  protoplast.  Up  to  the  time 
when  the  conjugation-pore  is  closed  the  antheridial  cell- wall  has  been 
of  the  same  thickness  as  that  of  its  stalk  cell  and  of  the  oogonium,  and 
has  showed  no  tendency  to  stain  differently  than  the  walls  of  these  cells. 
With  the  triple  stain  the  walls  in  these  stages  tend  to  stain  pale  blue. 
Very  soon  after  the  fusion  pore  is  closed,  however,  the  antheridial 
cell-wall  begins  to  increase  very  markedly  in  thickness  by  swelling  and 
undergoing  what  seems  probably  to  be  a  mucilaginous  degeneration 
(fig.  n).  This  swelling  is  all  toward  the  interior  of  the  cell.  With 
the  swelling  of  the  wall  the  protoplast  apparently  shrinks  together,  still 
keeping,  however,  the  general  outlines  of  the  cell.  The  surface  layer 
of  the  cell-wall  remains  dense  and  sharply  marked,  and  the  antheridial 
cell  as  a  whole  maintains  its  contour  quite  unchanged  or  with  a  very 
slight  sinking  in  of  the  walls,  indicating  diminished  turgidity. 

This  swelling  of  the  wall  continues  until  its  thickness  is  equal  to 
one-fourth  or  more  of  the  transverse  diameter  of  the  entire  antheridial 
cell  (figs.  12-20,  23,  24).  The  swelling  is  least  in  most  cases  on  the 
cross-wall  cutting  off  the  antheridium  from  its  stalk  cell  and  greatest 
on  its  outer  wall  opposite  the  region  in  which  it  is  pressed  against  the 


14  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

oogonium.  Commonly,  especially  in  the  earlier  stages,  the  thickening  is 
markedly  less  in  the  region  of  the  closed  conjugation-pore  (fig.  13). 
This  swollen  wall,  in  preparations  colored  with  the  triple  stain,  shows 
a  most  pronounced  affinity  for  the  orange.  In  preparations  given  an 
exposure  of  only  a  few  seconds  to  the  orange  this  wall  appears  deeply 
and  brightly  orange-yellow  in  color,  and  forms  a  most  conspicuous  and 
sharply  contrasted  object  in  sections  of  all  the  younger  stages  of  the 
ascocarp.  The  remains  of  the  protoplast  at  first  shrink  to  an  irregular 
oblong  body  which,  with  the  triple  stain,  usually  appears  red  and  is 
dense  and  structureless.  Later  this  mass  seems  to  grow  less  dense  and 
loses  its  staining  capacity,  so  that  ultimately  the  cavity  of  the  anther- 
idium  appears  quite  empty,  while  it  is  still  surrounded  by  the  conspic- 
uous swollen  wall. 

The  degeneration  of  the  remains  of  the  antheridial  cytoplasm  is 
an  interesting  process  in  itself,  and  a  comparison  of  its  structure  during 
the  successive  changes  which  it  undergoes,  with  the  structure  of  the 
adjacent  perithecial  cells,  is  interesting  in  several  points.  The  most 
conspicuous  change  which  it  seems  to  undergo  is  associated  with  an 
apparent  loss  of  water.  The  cytoplasm  of  the  perithecial  cells  is  loose 
and  spongy  in  texture,  with  large  spaces  filled  with  watery  cell-sap, 
while  the  non-stainable  cell-sap  seems  to  disappear  gradually  from  the 
antheridial  cytoplasm,  allowing  the  denser  portions  of  the  meshwork 
to  draw  together  so  as  to  form  ultimately  a  homogeneous  mass.  Fur- 
ther, while  the  normal  cytoplasm  tends,  with  the  triple  stain,  to  be 
colored  faintly  gray-blue,  or  slightly  buffy  with  longer  exposure  to  the 
orange,  the  contracted  cytoplasm  of  the  antheridial  cell  shows  a  pro- 
nounced affinity  for  the  safranin,  and  when  once  stained  red  retains 
the  color  with  great  tenacity,  so  that  the  entire  content  of  adjacent  cells 
may  be  decolorized  by  washing  in  alcohol  without  noticeably  affecting 
the  appearance  of  the  degenerating  antheridium.  The  change  in  the 
protoplast  here  is  similar  in  most  points  to  that  which  I  have  already 
described  (39)  as  taking  place  in  the  end  cell  of  the  promycelium  of 
the  anther-smut  after  the  two  basal  cells  have  been  joined  by  fusion- 
tubes.  The  end  cell  ordinarily  dies  under  these  conditions  and  its  pro- 
toplast forms  a  dense  red-stained  mass  like  that  of  the  antheridial  cell, 
except  that  in  the  promycelial  cell  the  nucleus  can  be  distinguished  for 
a  considerable  period,  while  in  the  degenerating  antheridial  cytoplasm 
no  nucleus  is  present.  The  swollen  yellow-stained  walls  of  the  anther- 
idial cell  makes  it  a  conspicuous  object  in  sections  of  the  perithecium 
in  all  its  earlier  development.  The  empty  wall  persists  until  the  peri- 
thecium is  half-grown,  occupying  a  characteristic  position  toward  the 


DEVELOPMENT    OF    THE    PERITHECIUM.  15 

base  of  the  perithecium  considerably  to  one  side  of  its  stalk  and  com- 
monly in  about  the  third  layer  of  cells  from  the  surface  of  the  perithe- 
cial  wall.  From  the  time  of  the  completion  of  the  process  of  fertilization 
and  the  closure  of  the  conjugation-pore  it  is  most  conspicuously  a  dead 
cell,  and  its  differentiation  from  all  the  later-formed  perithecial  protect- 
ive cells,  both  in  function,  structure,  and  fate,  is  one  of  the  most  con- 
spicuous facts  that  appears  in  any  series  of  sections  of  the  developing 
ascocarp  of  Phyllactinia,  and  is  in  itself  sufficient  to  suggest  the  mor- 
phological relationships  of  the  ascocarp. 

The  fertilized  oogonium  in  Phyllactinia  begins  its  further  devel- 
opment at  once.  The  egg  in  this  case  is  the  oogonium,  and  the  use  of 
the  term  oosphere  seems  quite  superfluous.  We  have  no  rounding  up 
of  the  protoplast  of  the  oogonium  and  separation  from  its  wall  to  form 
an  oosphere,  as  in  Oedogonium  or  Vaucheria.  Still,  there  is  no  ques- 
tion that  the  female  cell  in  Phyllactinia  corresponds  entirely  to  that  of 
Oedogonium ;  and  the  fact  that  it  does  not  round  itself  up  is  due  to  its 
habit  of  continued  development  as  a  cell  of  the  mycelium  which  pro- 
duced it.  It  is  set  free  from  vegetative  continuity  with  the  mother 
plant  in  no  respect.  Whether  it  is  to  be  regarded  as  the  beginning  of 
a  new  individual  life  history  is  a  question  which  must  be  settled  on 
other  grounds  than  that  of  its  being  set  free  from  the  mother  plant  as 
an  independent  life  unit. 

While  the  process  of  fertilization  is  going  on,  the  protective  branches 
which  are  to  form  the  perithecial  envelope  push  out  from  the  stalk-cell 
of  the  oogonium  and  grow  up  about  the  two  gametephores,  applying 
themselves  closely  to  their  surface  and  following  an  irregular  path, 
owing  to  the  slight  spiral  twisting  of  the  oogonium.  In  the  figures  I 
have  put  in  only  such  portions  of  them  as  lay  outside  the  outlines  of  the 
sexual  apparatus.  In  Phyllactinia  it  is  very  conspicuous  that  these 
enveloping  branches  may  and  do  arise  from  the  stalk-cell  of  the  anther- 
idium  as  well  as  from  that  of  the  oogonium  (figs.  14,  17).  Even 
before  fertilization  is  complete  the  antheridial  stalk-cell  becomes  much 
enlarged  and  is  apparently  in  a  very  active  vegetative  condition  (fig.  7), 
resembling  that  of  the  stalk-cell  of  the  oogonium.  After  fertilization 
its  condition  is  in  striking  contrast  with  that  of  the  antheridial  cell  which 
was  cut  off  from  it.  While  the  walls  of  the  latter  swell  and  apparently 
become  mucilaginous,  and  its  protoplast  degenerates  as  described  above, 
the  stalk-cell  continues  to  enlarge,  its  nucleus  divides,  and  hyphal  buds 
push  out  from  its  surface  At  the  very  first  one  or  more  hyphal  branches 
push  out  just  below  the  cross- wall  separating  the  antheridium  from  the 
stalk-cell  and  grow  up  over  the  former  (fig.  14),  inclosing  and  covering 


1 6          SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

it  and  thus  closing  the  apex  of  the  ascocarp,  though,  as  noted,  because 
of  the  curved  form  of  the  oogonium,  this  region  may  not  be  vertically 
over  the  stalk-cell  of  the  oogonium.  The  further  growth  of  these  and 
other  hyphae  from  the  antheridial  stalk-cell  plays  an  important  part  in 
the  development  of  the  perithecial  envelopes,  but  the  branches  from  the 
oogonial  stalk  and  those  from  the  antheridial  stalk  become  so  inter- 
mingled that  it  is  not  easy  to  distinguish  them  in  later  stages. 

While  the  first  enveloping  branches  are  pushing  up  from  the  stalk- 
cells  the  oogonium  is  also  beginning  its  further  development.  From 
its  function  in  the  mature  ascocarp  we  may  call  this  product  of  the 
growth  of  the  oogonium  the  ascogonium.  This  growth  of  the  oogonium 
is  essentially,  as  noted,  the  germination  of  a  fertilized  egg  which,  instead 
of  being  set  free  from  the  mother  plant,  continues  in  unaltered  vegeta- 
tive connection  with  it.  In  its  growth  the  ascogonium  elongates  and 
increases  in  diameter.  At  first,  since  the  antheridial  stalk-cell  does  not 
elongate  and  the  end  of  the  ascogonium  is  firmly  attached  to  the  anther- 
idium,  the  two  ends  of  the  ascogonium  are  held  at  about  the  same  dis- 
tance apart  as  they  were  at  the  time  of  fertilization,  and  its  growth 
results  in  a  bulging  out  and  increased  convexity  of  its  free  middle  por- 
tion. The  suggestion  given  by  sections  of  the  growing  fruits  at  this 
time  is  that  of  a  mutual  rivalry  in  growth  between  the  ascogonium  and 
the  enveloping  hyphae.  The  ascogonium  tends  constantly  to  burst  out 
of  the  inclosing  hyphae,  which  latter,  by  elongation  and  richly  branch- 
ing, tend  to  inclose  and  cover  it  over  its  whole  surface. 

Whether  or  not  the  inclosing  hyphae  grow  more  slowly  at  first  and 
actually  exert  pressure  on  the  growing  ascogonium,  it  is  certain  that 
the  ascogonium  becomes  curved  in  a  very  irregular  fashion  at  this 
stage.  The  sections  (figs.  12-23)  are  about  5  /*  thick,  and  the  asco- 
gonium bends  up  and  down  in  the  thickness  of  the  section  in  a  fashion 
which  would  be  difficult  to  represent  without  special  shading,  which 
would  interfere  with  the  representation  of  its  protoplasmic  content,  etc. 
Hence,  I  have  here,  as  in  the  earlier  stages,  disregarded  in  the  drawings 
the  vertical  bending  of  the  ascogonium,  and  have,  by  focusing  up  and 
down,  shown  it  in  median  optical  section  as  if  projected  in  the  plane  of 
the  section.  This  has  resulted,  of  course,  in  making  it  appear  some- 
what shorter  than  it  really  is,  and  as  a  further  result,  in  cases  of  suc- 
cessive sections  in  which  the  ascogonium  appears,  its  parts  do  not  seem 
to  fit  together  as  they  would  if  its  actual  windings  were  shown  in  the 
figures.  Since,  however,  we  are  concerned  only  with  the  facts  of  its 
increased  size,  the  multiplication  of  its  nuclei,  its  division  into  cells,  and 


DEVELOPMENT    OF   THE    PERITHECIUM.  I/ 

ultimate  branching  to  form  the  ascogenous  hyphse,  the  representation  of 
its  exact  form  may  be  considered  as  a  matter  of  secondary  importance. 

With  the  first  growth  of  the  ascogonium  in  size  the  fusion  nucleus 
divides  and  we  have  a  binucleated  stage,  which  is  apparently  rather 
long  continued,  lasting  until  the  complete  inclosure  of  the  ascogonium 
by  the  enveloping  hyphse  (figs.  13-16).  It  is  an  apparently  abundant 
stage  and  an  easy  one  to  study,  since  the  fruits  are  now  large  enough 
to  be  readily  found,  and  the  greater  part  of  the  ascogonium  and  the  old 
antheridial  cell  can  frequently  be  found  in  a  single  section.  The  nuclei 
are  as  a  rule  symmetrically  placed  in  the  axis  of  the  ascogonium.  In 
Phyllactinia,  according  to  my  observations,  cell  division  never  follows 
this  first  division  of  the  egg-nucleus.  The  ascogonium  remains  one- 
celled  and  its  nuclei  continue  to  divide.  As  to  how  many  nuclear 
divisions  may  precede  cell  division  I  am  not  certain,  but  in  the  end  we 
have  formed  a  row  of  from  3  to  5  cells  (figs.  17-22).  The  end  cell  of 
the  ascogonium  regularly  contains  one  nucleus  and  remains  attached 
to  the  old  antheridial  cell  for  a  considerable  period,  though  finally  the 
two  are  commonly  forced  apart,  apparently  by  pressure  of  the  surround- 
ing cells  of  the  enveloping  hyphse.  Sometimes  this  separation  may 
occur  at  quite  an  early  period  (fig.  i8&).  The  old  antheridial  cell  com- 
monly remains  at  a  depth  of  about  two  or  three  cell  layers  from  the 
surface  of  the  perithecium,  while  the  end  of  the  ascogonium  may 
become  more  deeply  buried  by  growth  of  the  perithecial  hyphse  around 
it.  The  ascogonium  forms  at  its  maturity  a  single  row  of  cells,  the 
penultimate  one  of  which  regularly  contains  more  than  one  nucleus. 

The  next  step  in  the  development  of  the  ascocarp  consists  in  the 
formation  of  the  so-called  ascogenous  hyphse.  These  arise  as  lateral 
branches  from  the  ascogonium.  Whether  they  all  arise  from  a  single 
cell  of  the  ascogonium,  as  one  might  expect  from  analogy  with  Asco- 
bolus,  or  whether  two  or  three  of  the  upper  cells  may  sprout  out  in 
branches,  is  not  easy  to  determine.  It  is  certain  that  some  branches 
arise  from  the  penultimate  cell.  The  ascogenous  hyphse  arise  relatively 
early  in  the  development  of  the  ascocarp  in  Phyllactinia  (figs.  22,  250) 
at  a  time  when  the  ascogonium  is  inclosed  by  only  about  two  layers  of 
perithecial  cells.  It  is  thus  impossible  for  them  to  grow  out  vertically 
from  the  ascogonium  for  any  great  distance,  and  the  result  is  that  they 
lie  flat  on  the  cells  of  the  ascogonium,  crowding  back  the  perithecial 
cells  and  overlapping  and  intertwining  with  each  other  so  as  to  cover 
the  whole  upper  part  of  the  ascogonium.  This  makes  it  very  difficult 
to  trace  a  particular  branch  to  its  point  of  origin,  especially  since  the 
walls  of  the  cells  are  thin  and  their  form  distorted  by  the  crowding  of 


l8  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

the  perithecial  cells  and  the  young  ascogenous  hyphse.  The  questions 
here  involved  are  of  interest  as  bearing  on  the  relationship  of  the  mil- 
dews with  such  forms  as  Ascobolus  and  Pyronema,  but  have  no  direct 
bearing  on  our  interpretation  of  the  ascogonium  or  the  entire  ascocarp. 

In  view  of  Blackman's  and  Christman's  remarkable  discoveries  in 
the  rusts,  noted  further  below,  I  have  devoted  special  attention  to  the 
question  as  to  whether  in  Phyllactinia,  either  in  the  ascogonium  or  the 
ascogenous  hyphae,  binucleated  cells  may  not  be  present.  My  results 
have  been  entirely  negative.  While  occasionally  a  binucleated  cell  is 
found  (fig.  210)  in  the  ascogonium,  it  is  even  here  uncertain  whether 
a  subsequent  cell  division  may  not  separate  these  nuclei.  The  great 
majority  of  the  cells  are  uninucleated.  As  just  noted,  the  ascogenous 
hyphae  in  Phyllactinia,  in  whole  or  in  large  part,  arise  from  the  penulti- 
mate cell  of  the  ascogonium,  and  this  cell  regularly  contains  several 
nuclei  (figs.  19,  20,  21,  253).  The  cells  that  are  to  become  asci  contain 
two  nuclei  (figs.  28,  29).  The  remaining  cells  of  both  the  ascogonium 
and  the  ascogenous  hyphae  after  cell  division  is  complete  are  almost 
without  exception  uninucleated  (fig.  19-29).  This  comes  out  very 
strikingly  in  the  case  of  the  old  cells  of  the  ascogonium  and  ascogenous 
hyphae,  which  are  largely  filled  with  a  watery  cell-sap  (figs.  2$b,  29). 

The  enveloping  hyphae  of  the  perithecium  continue  to  grow,  and 
thus  the  protective  envelope  is  enlarged.  The  ascogonium  reaches 
its  full  development  rather  early  in  forming  the  row  of  cells  above 
described,  and  then  ceases  to  enlarge  and  remains  lying  in  the  basal 
portion  of  the  growing  perithecium  (fig.  27).  With  the  enlargement 
of  the  perithecium  the  ascogenous  hyphae  push  up  vertically  and  away 
from  the  ascogonium.  It  is  quite  plain  from  fig.  27,  as  also  from  a 
study  of  the  earlier  stages,  that  the  ascogenous  hyphae  develop  consid- 
erably before  they  become  septate.  Later,  after  cell  division  is  com- 
plete, the  asci  are  formed,  either  as  lateral  outgrowths  from  intercalary 
cells  or  from  the  terminal  cell  of  an  ascogenous  hypha.  In  fig.  29^  we 
find  the  terminal  cell  developing  as  an  ascus  and  also  evidence  of  the 
repeated  pushing  out  of  the  subterminal  cells,  though  it  is  possible  that 
this  appearance  is  due  to  the  formation  of  the  entire  branch  before  cell 
division  occurred.  In  this  figure  the  second  cell  appears  empty  of  con- 
tents, but  this  is  due  to  the  fact  that  only  a  small  portion  of  its  base 
appeared  in  the  section  drawn,  the  remaining  portion  extending  into 
the  next  section.  In  this  case  the  two  cells  which  are  to  produce  asci  are 
each  binucleated,  while  the  stalk-cells  below  are  uninucleated.  Quite 
possibly  the  cell  division  does  not  occur  in  the  ascogenous  hyphae  until 
the  stage  when  the  young  asci  are  differentiated,  and  this  gives  full 


DEVELOPMENT    OF   THE    PERITHECIUM.  IQ 

possibility  for  special  arrangements  of  the  nuclei  as  to  their  distribution 
in  asci  and  stalk-cells.  Fig.  290,  shows  an  ascogenous  hypha  in  which 
the  end  cell  is  enlarged  and  is  apparently  destined  to  form  an  ascus,  but 
contains  only  a  single  nucleus.  I  am  inclined  to  think  this  is  due  to 
the  fact  that  the  cell  in  question  is  cut  in  two  and  that  a  portion  of  it 
should  appear  in  the  next  section.  In  this  particular  case,  however,  I 
have  been  unable  to  find  the  second  nucleus,  and  it  is  possible  that  we 
have  here  a  case  of  a  young  ascus  just  cut  off  and  containing  only  one 
nucleus.  If  this  is  the  fact  it  is  certainly  to  be  regarded  as  a  rare  excep- 
tion to  the  rule  that  the  young  ascus  contains  two  nuclei  when  first 
formed.  The  cell  below  this  in  the  same  hypha  contains  two  nuclei 
and  is  pushing  out  laterally  and  upward  to  form  an  ascus  in  the  normal 
fashion.  The  third  cell  is  plainly,  from  its  lack  of  content,  to  remain 
sterile  and  contains  a  single,  much  smaller  nucleus.  It  is  evident  that 
there  is  in  Phyllactinia,  as  also  in  Erysiphe,  no  such  regular  process  of 
forming  an  ascus  from  the  penultimate  cell  of  a  curved  lateral  branch 
of  an  ascogenous  hypha  as  one  finds  in  Ascobolus,  Pustularia,  Pyro- 
nema,  and  some  other  Discomycetes. 

The  young  ascus  regularly  contains  two  nuclei,  and  since,  as  noted, 
the  ascogenous  hyphae  are  multinucleated  before  they  are  cut  up  into 
cells  and  their  cells,  which  are  to  form  asci,  regularly  contain  two  nuclei 
from  the  time  they  are  formed,  there  is  no  reason  for  supposing  that 
the  pairs  of  nuclei  are  daughter  nuclei  of  the  same  mother  nucleus ;  but 
there  is  no  such  apparent  method  of  preventing  the  inclusion  of  two 
sister  nuclei  in  the  same  ascus  as  one  finds  in  the  Discomycetes  men- 
tioned above.  Certain  cells  of  the  ascogenous  hyphse  which  remain 
sterile  contain  only  one  nucleus,  and  this  may  be  the  reason  for  their 
failure  to  develop  further.  Still  other  cells  of  the  ascogenous  hyphse 
containing  two  nuclei  are  apparently  prevented  from  developing  by 
overcrowding,  so  that  one  can  not  conclude  positively  that  the  number 
of  nuclei  which  it  contains  determines  necessarily  the  fate  of  the  cell 
of  an  ascogenous  hypha.  On  the  analogy  with  the  Discomycetes  it 
seems  fair  to  assume,  in  the  absence  of  any  evidence  to  the  contrary, 
that  the  two  nuclei  in  the  young  ascus  are  not  sister  nuclei,  though 
there  is  again  no  reason  for  assuming  in  Phyllactinia  that  their  relation- 
ship is  a  very  distant  one.  The  asci  now  elongate  rapidly  in  a  vertical 
direction,  while  the  sterile  cells  of  the  ascogenous  hyphae  with  which 
they  are  connected  below  undergo  no  further  development.  As  a  result 
these  sterile  cells  and  the  old  ascogonium  come  to  lie  further  from  the 
center  of  the  perithecium  and  are  more  noticeably  left  behind,  as  it  were, 
in  the  basal  portion.  The  asci,  on  the  contrary,  continue  to  occupy  a 


2O  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

central  position.  Their  shape  at  this  time  is  well  shown  in  fig.  41.  It 
is  noticeable  that  they  are  thicker  in  their  upper  portion  and  taper  con- 
siderably below.  A  comparison  of  figs.  53-56  with  fig.  41  suggests  that 
the  vertical  elongation  of  the  asci  has  been  mainly  in  this  lower  tapering 
region,  which  has  been  extended  as  a  stalk-like  prolongation  connecting 
the  upper  thickened  portion,  in  which — it  is  to  be  noted — lie  the  nuclei, 
with  the  sterile  cells  of  the  ascogenous  hyphae  below.  This  tapering 
stalk  portion  is  never  cut  off  by  further  cross-walls  to  form  a  true  stalk- 
cell.  A  considerable  portion  of  it  thickens  up  later  and  the  nucleus 
comes  to  lie  much  further  down  than  in  the  present  stage.  There  is 
always,  however,  in  the  mature  asci  an  abruptly  narrowed  basal  portion 
(see  figs.  55,  56)  where  the  ascus  connects  with  the  next  adjacent  cell  of 
the  ascogenous  hypha.  A  basal  stalk-cell  is,  however,  never  cut  off 
from  the  lower  end  of  the  ascus.  It  remains  always  the  entire  cell  of 
the  ascogenous  hypha,  as  it  was  formed  when  the  hyphae  became  septate. 

The  young  asci  are  very  closely  inclosed  from  the  start  by  the 
vegetative  hyphse  of  the  perithecium.  At  a  time  when  the  asci  are 
scarcely  differentiated  and  the  division  of  the  ascogenous  hyphse  into 
binucleated  cells  is  scarcely  complete  the  perithecium  has  the  general 
structure  shown  in  fig.  27.  No  differentiation  of  the  perithecium  into 
separate  layers  can  be  made  out  at  this  stage.  There  is  no  evidence  of 
a  radial  growth  of  hyphal  branches  inward  from  the  perithecial  wall. 
The  hyphae  of  the  perithecial  wall  extend  between  the  branches  of  the 
ascogenous  hyphaa  so  that  the  latter  hardly  come  in  contact  with  each 
other,  but  are  separated  and  partly  inclosed  by  hyphal  branches  which, 
from  their  subsequent  development  and  differentiation,  we  may  sup- 
pose— even  at  this  early  stage — are  nurse  cells  which  give  up  food 
stuffs  to  the  ascogenous  cells  among  which  they  lie  and  which  are  des- 
tined to  perform  the  spore-bearing  function. 

Median  sections  show  the  cells  of  the  perithecium  for  the  most  part 
as  oblong  in  section  and  apparently  uninucleated.  Tangential  sections 
show,  however,  that  at  this  stage  they  are  in  reality  flattened  polygonal 
plates  and  contain  generally  three,  four,  or  even  more  nuclei.  The 
original  stalk-cell  of  the  oogonium  is  shown  in  figs.  22  and  27  forming 
the  center  of  support  and  attachment  for  the  entire  perithecium.  The 
lower  cells  of  the  ascogonium  extend  upward  from  this  stalk-cell  and 
curve  to  one  side  in  the  base  of  the  ascocarp.  These  cells  are  large  and 
swollen  in  appearance.  The  ascogonium  as  a  whole  is  never  found 
lying  in  a  single  plane  so  as  to  appear  entire  in  a  single  section.  Gen- 
erally its  path  is  so  irregular  and  it  has  become  so  distorted  by  the  press- 
ure of  the  perithecial  cells  that  its  successive  cells  bend  back  and  forth 


DEVELOPMENT    OF   THE    PERITHECIUM.  21 

in  a  fashion  very  difficult  to  show  in  even  a  large  drawing.  Drawing 
the  successive  cells  as  they  appear  in  one  plane,  as  I  have  done,  results, 
for  example,  in  causing  the  second  cell  of  the  ascogonium  in  fig.  27  to 
appear  very  short  as  compared  with  the  first  and  third.  As  a  matter 
of  fact,  however,  the  second  cell  is  as  large  as  the  first,  but  bulges  out 
on  the  lower  side,  as  the  section  lies,  so  as  to  appear  for  the  most  part 
in  the  next  section. 

From  the  outer  cells  of  the  perithecium  in  its  basal  region  hyphae 
have  begun  to  sprout,  which  form  a  sort  of  secondary  mycelium  com- 
parable to  the  secondary  mycelium  of  many  Discomycetes  (fig.  27). 
These  hyphae  grow  out  for  a  considerable  distance  and  become  inter- 
woven with  the  mycelial  hyphse  over  the  stomata.  Whether  they  give 
rise  to  internal  hyphal  branches  with  haustoria  I  have  been  unable  to 
determine  with  certainty,  as  they  are  in  no  way  differentiated  from  ordi- 
nary mycelial  hyphae  in  appearance  and  their  course  is  not  easy  to  trace. 
There  are  good  reasons,  however,  for  doubting  whether  they  ever  pass 
through  the  stomata.  The  latter  are  crowded  full,  with  all  the  entering 
branches  which  apparently  can  find  room,  long  before  the  perithecia 
are  sufficiently  developed  to  give  rise  to  these  secondary  hyphae.  It 
seems  likely  that  the  latter  serve  merely  for  the  better  attachment  and 
support  of  the  developing  perithecium.  The  single  stalk-cells  of  the 
oogonium  and  antheridium  together  certainly  seem  hardly  adequate, 
from  a  mechanical  point  of  view,  for  giving  a  firm  and  safe  support 
for  the  relatively  immense  fruit  body  developed  on  them.  In  a  section 
such  as  that  shown  in  fig.  30  the  asci  seem  already  to  lie  at  approxi- 
mately the  same  level  in  the  ascocarp,  and  to  be — for  the  most  part,  at 
least — in  a  horizontal  layer  slightly  above  the  middle  of  the  perithecium. 
Close  examination  and  comparison  of  successive  sections  show,  how- 
ever, that  in  reality  the  individual  asci  extend  to  quite  unequal  levels 
both  toward  the  base  and  toward  the  apex  of  the  perithecium,  as  would 
be  expected  from  the  irregular  course  of  the  ascogenous  hyphae  from 
which  they  arise.  At  this  stage  the  two  nuclei  of  the  young  ascus  have 
fused  to  form  the  primary  ascus-nucleus  of  De  Bary  and  Strasburger. 
The  process  of  fusion  is  described  fully  below. 

In  this  stage  of  the  development  of  the  perithecium  the  old  asco- 
gonium and  the  sterile  cells  of  the  ascogenous  hyphae  are  scarcely  rec- 
ognizable. The  asci  have  grown  to  be  swollen,  oblong  sacks  and  are 
pressed  together  and  flattened  upon  each  other  in  their  middle  regions, 
while  the  perithecial  cells  still  press  in  between  their  ends.  The  nucleus 
of  each  ascus  lies  in  its  lower  end  and  below  this  the  ascus  is  narrowed 
sharply  into  a  stalk.  This  stalk-like  portion  frequently  does  not  lie  in 


22  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

the  same  plane  as  the  body  of  the  ascus,  and  hence  does  not  appear  in 
the  section  in  which  the  body  of  the  ascus  is  best  shown.  In  fig.  30  the 
second  and  third  asci,  counting  from  the  right,  show  the  narrowed 
basal  portion,  and,  as  suggested,  these  figures  do  not  show  the  body  of 
the  ascus  so  fully  as  those  lying  further  to  the  left. 

At  this  stage  the  wall  of  the  perithecium  in  Phyllactinia  can  be 
roughly  differentiated  into  three  layers.  The  outermost  consists  of  a 
single  layer  of  peripheral  cells.  From  these  cells  in  the  basal  region 
the  secondary  mycelial  hyphse  sprout  as  noted  above.  From  this  same 
layer  in  the  equatorial  region  of  the  perithecium  the  spine-like  append- 
ages with  enlarged  bulbous  bases  arise.  These  have  not  yet  begun  to 
develop  at  the  stage  shown  in  fig.  30.  From  the  same  peripheral  layer, 
in  a  zone  about  the  apical  region  of  the  perithecium,  are  developed  the 
peculiar  penicillate  cells  which  have  been  so  frequently  the  objects  of 
study  in  recent  years.  The  first  beginnings  of  these  outgrowths  are 
shown  in  the  swollen  and  protuberant  form  of  certain  of  the  peripheral 
cells  in  the  upper  portion  of  fig.  30,  to  the  left.  The  outer  layer  of 
perithecial  cells  in  Phyllactinia  is  especially  active  in  growth.  They 
remain  thin-walled  and  show  no  tendency  to  become  hardened  and  to 
dry  out  until  the  perithecium  is  nearly  ripe. 

Beneath  the  peripheral  layer  is  a  zone  several  cells  in  thickness, 
which  functions  as  a  protective,  mechanical,  strengthening  layer  from 
a  relatively  early  stage  in  the  development  of  the  perithecium.  Its 
protoplasts  contain  large  vacuoles  and  its  cell-walls  undergo  a  change 
apparently  analogous  to  lignification.  The  cells  appear  as  poor  in  con- 
tent and  with  specially  strengthened  walls.  Inside  this  zone  and  next 
to  the  developing  asci  are  several  layers  of  cells  richly  filled  with  proto- 
plasm and  with  thin,  apparently  unmodified  walls.  These  cells  consti- 
tute a  close  packing  about  the  developing  asci  and,  as  has  been  suggested, 
are  very  probably  concerned  with  their  nutrition,  though  it  is  difficult 
to  bring  positive  evidence  on  this  point. 

The  later  development  of  the  perithecial  walls  consists  simply  in 
further  differentiation  of  the  parts  already  indicated.  The  penicillate 
cells  already  referred  to  are  very  easily  studied  in  all  stages  of  their 
development.  They  arise,  as  noted,  as  blunt  outgrowths  of  the  periph- 
eral cells  in  a  zone  about  the  apex  of  the  perithecium.  They  are  either 
not  developed  at  all,  or  are  relatively  small  in  the  apical  region  itself. 
The  cells  which  give  rise  to  them  push  up  vertically  to  the  surface  of 
the  host-leaf,  rather  than  radially  to  the  surface  of  the  perithecium. 
They  are  at  first  blunt  and  unbranched  projections  from  the  peripheral 
cells  (fig.  50),  but  after  they  have  reached  a  height  equal  to  about  twice 


DEVELOPMENT    OF   THE    PERITHECIUM.  23 

or  three  times  their  diameter  they  suddenly  break  up  into  a  number  of 
very  fine,  thread-like  branches,  which  bud  out  from  their  upper  ends 
and  grow  on  up  to  a  height  about  equal  to  that  of  the  unbranched  basal 
portion  of  the  cell.  This  is  the  simplest  type  of  these  cells  in  the  forms 
of  Phyllactinia  I  have  studied.  Much  more  abundant  are  other  types 
which,  before  breaking  up  into  the  ultimate  thread-like  branches,  divide 
first  into  two  or  three  main  branches,  which  may  be  very  unequal  or 
approximately  equal  in  size  (fig.  51).  As  a  rule  this  latter  type  of 
cells  lies  farther  away  from  the  apex  of  the  perithecium,  and  hence, 
owing  to  its  curved  surface,  must  grow  higher  in  order  to  bring  the 
ends  of  the  filamentous  branches  to  a  level  with  those  of  the  more  cen- 
trally placed  brush  cells.  The  penicillate  cells  begin  their  development 
some  time  after  the  fusion  of  the  nuclei  in  the  ascus  is  complete,  and 
have  completed  their  growth,  as  a  rule,  before  the  nucleus  of  the  ascus 
begins  to  divide  preparatory  to  spore  formation.  The  penicillate  cell  is 
never  divided,  but  remains  throughout  simply  an  enlarged  and  branched 
peripheral  cell  of  the  perithecium.  It  contains  two  or  three  nuclei,  as 
a  rule,  before  its  special  growth  begins,  and  when  fully  developed  may 
contain  as  many  as  eight  or  ten  nuclei.  These  are  always  situated  in 
the  enlarged  basal  portion  of  the  cell  and  never  penetrate  into  the  fila- 
mentous branches.  They  form  generally  an  irregular,  scattered  group 
in  the  upper  part  of  the  basal  portion  of  the  cell  (fig.  51) . 

As  soon  as,  or  even  before,  the  penicillate  cell  reaches  its  full  size 
the  walls  of  the  filamentous  branches  begin  to  swell  and  become  gelati- 
nous. As  a  result  they  become  fused  together  laterally  to  form  the 
slimy  mass  crowning  the  perithecium  which  has  been  described  by  many 
authors.  The  wall  and  contents  of  the  basal  portion  of  the  cells  remain 
unchanged  for  some  time  (fig.  51),  but  gradually  it,  too,  is  more  or  less 
involved  in  the  degenerative  processes,  and  almost  the  entire  walls  and 
contents  of  the  penicillate  cells  are  ultimately  converted  into  a  sticky, 
gelatinous  mass. 

When  the  walls  of  the  filamentous  branches  swell,  their  cell  content 
is  reduced  to  a  mere  granular  thread  except  at  the  very  apex,  though 
earlier  a  well-marked  prolongation  of  the  protoplast  with  normal  proto- 
plasmic appearance  extended  to  the  end  of  each  branch.  With  the 
swelling  of  the  wall  this  structure  gradually  deteriorates,  though  a  gran- 
ular thread  persists  till  a  late  stage  to  mark  the  original  lumen  of  the 
branch.  At  the  very  apex  the  wall  does  not  swell  so  strongly  and  the 
protoplast  also  persists  as  a  small  oval  vesicle  tapering  below  into  the 
granular  thread  just  mentioned  (fig.  51).  As  a  result  the  upper  ends 
of  the  filaments  are  slightly  enlarged  and  the  surface  of  the  slime  mass 


24  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

is  covered  by  a  layer  of  these  oval  vesicles.  It  is  possible  that  the  vesicles 
burst  and  exude  a  slime  more  liquid  than  that  of  the  deliquescent  walls 
at  the  time  when  the  perithecium  is  overturned,  and  thus  aid  in  making 
more  certain  that  it  shall  stick  fast  to  any  object  which  touches  it.  In 
this  gelatinous  cap,  when  the  perithecia  are  ripe,  the  hyphae  of  molds 
may  frequently  be  found  growing.  The  functions  and  adaptations  of 
this  slime-cap  have  been  fully  discussed  by  Neger  (68),  and  I  need 
not  go  into  the  matter  further  here. 

The  appendages  arise  in  a  zone  about  the  equatorial  region  of  the 
perithecium  as  swollen  cells  of  the  peripheral  layer.  As  has  been  many 
times  described,  in  their  mature  condition  they  consist  of  a  slender, 
straight  spine  with  a  very  much  enlarged  bulbous  base.  They  grow 
horizontally  outward  in  a  bristling  phalanx  about  the  middle  of  the 
fruit  body.  As  it  ripens  they  bend  downward  and,  pressing  on  the  leaf 
surface,  lift  the  fruit  up,  tearing  it  loose  from  its  stalk  and  the  second- 
ary mycelial  filaments  described  above.  It  is  thus  set  free  to  fall  or  be 
blown  by  the  wind  until  it  strikes  and  adheres  to  some  object  by  the 
mucilaginous  cap  described  above.  As  a  matter  of  fact,  what  seems 
frequently  to  happen  is  that  the  perithecium  simply  rolls  over  and 
remains  sticking  wrong  side  up  on  the  same  leaf  on  which  it  grew. 

The  walls  of  the  appendages  very  early  become  hardened  and 
brittle,  so  that  they  break  up  in  cutting,  and  it  is  not  common  to  find 
good  entire  longitudinal  sections  of  them  in  microtome  preparations. 
Still,  their  structure  is  very  clearly  shown.  They  contain  an  apparently 
living  protoplast  until  late  in  the  ripening  of  the  perithecium.  As  a 
rule  they  have  only  one  or  two  nuclei.  It  is  quite  common  to  find  the 
single  nucleus  lying,  not  in  the  bulbous  base,  but  somewhere  in  the  basal 
region  of  the  spine.  The  cytoplasm  consists  of  a  thin  lining  layer  just 
inside  the  wall  and  large  vacuolar  cavities  filling  the  greater  part  of  the 
bulb  and  extending  into  the  spine.  The  thickening  of  the  wall  is  char- 
acteristic and  especially  adapted  for  producing  the  motions  of  the 
appendage,  as  noted  above.  The  spine  is  thick-walled  throughout,  its 
apex  being  without  a  lumen  for  some  little  distance.  The  bulb  is  thick- 
walled  over  its  upper  surface,  but  on  the  under  side  there  is  an  oval 
region  whose  wall  has  remained  thin.  This  thin  area  extends  up  on 
the  sides  of  the  bulb  also.  The  functioning  of  the  structure  so  formed 
is  very  simple.  As  the  perithecium  loses  water — dries  out  in  beginning 
to  ripen — the  appendage  loses  water  also  by  evaporation.  This  results 
in  a  pushing  in  by  atmospheric  pressure  of  the  thin  area  on  the  bottom 
of  the  bulb  and  a  consequent  pulling  down  of  the  end  of  the  spine  as 
a  result  of  the  shortening  of  the  under  surface  of  the  bulb.  If  the 


DEVELOPMENT    OF   THE    PERITHECIUM.  25 

appendage  is  allowed  to  absorb  moisture  again  it  will  straighten  out, 
and  by  drying  again  the  bending  may  be  repeated.  Neger  (68) 
describes  the  repetition  of  the  process  a  number  of  times  as  a  result  of 
alternately  moistening  and  drying  the  perithecia.  The  protoplast  is 
apparently  living  in  the  appendages  of  Phyllactinia  till  late  in  the  ripen- 
ing, at  least  till  after  they  have  performed  their  function  in  breaking 
the  perithecia  loose.  Neger's  (69)  experiments  on  old,  dead  perithecia, 
in  which  he  found  the  appendages  still  capable  of  executing  their 
hygroscopic  motions,  would  seem  to  show  that  the  living  protoplast 
is  not  essential  for  their  proper  functioning.  The  small  number  of 
nuclei  in  the  spinous  appendage  as  compared  with  that  in  the  actually 
much  smaller  penicillate  cell  is  notable  and  may  perhaps  be  connected 
with  the  difference  in  function  of  the  two  types  of  outgrowths.  The 
work  of  the  penicillate  cells  is  largely  chemical  in  the  formation  of  the 
mucilaginous  cap ;  that  of  the  spinous  appendages  is  largely  mechanical 
in  the  bending  motions  they  execute  in  tearing  the  perithecium  loose 
from  its  attachments. 

With  the  full  maturity  of  the  perithecium  the  development  of  the 
spores  in  the  asci  begins.  As  a  matter  of  fact,  in  sectioned  material  one 
almost  never  finds  spore  formation  beginning  until  after  the  perithecia 
have  broken  loose  from  their  original  position  and  are  lying  wrong  side 
up  and  attached  by  the  mucilaginous  cap  described  above.  It  is  pos- 
sible, of  course,  that  many  perithecia  may  be  overturned  prematurely 
in  the  processes  of  fixing,  etc.  Still  only  those  in  which  the  brush  cells 
have  formed  their  mucilage  will  become  fixed  to  the  leaf  and  so  appear 
in  the  sections.  The  number  of  asci  in  a  perithecium  varies  from  12  to 
25.  Median  sections  through  the  perithecium  show  sections  of  from 
3  to  5  or  6  asci.  The  bursting  of  the  perithecia  and  asci  and  the  germi- 
nation of  the  ascopores  have  not  been  observed  for  Phyllactinia,  so  far 
as  I  am  aware. 


26  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 


MORPHOLOGY   OF  THE  ASCOCARP. 

Considerable  further  evidence  that  the  ascocarp  originates  in  a 
functional  sexual  apparatus  has  accumulated  in  recent  years.  Miss 
Dale  (18)  has  shown  conclusively  that  a  fusion  of  gametes  occurs  at  the 
origin  of  the  ascocarps  of  Gymnoascus  Reesii  and  Gymnoascus  candi- 
dus,  and  concludes  that  the  normal  method  of  origin  of  the  fruit  bodies 
of  the  Gymnoascaceae  and  their  related  forms  is  by  the  union  of  two 
cells  of  more  or  less  widely  separated  origin.  Monascus  is  a  very  inter- 
esting object  of  investigation  and  its  affinities  have  been  quite  variously 
interpreted.  Barker's  (3)  complete  and  consistent  account  and  the 
more  recent  briefer  one  by  Olive  (72)  agree  in  describing  the  origin  of 
the  asci  from  ascogenous  hyphse  and  an  ascogonium,  so  that  we  may 
probably  conclude  that  it  belongs  among  the  Ascomycetes.  Barker, 
Ikeno,  and  Olive  maintain  the  existence  of  a  sexual  cell-fusion,  while 
Kuyper  (55,  56)  and  Dangeard  (20)  deny  that  any  such  process  occurs. 

Ikeno's  account  of  two  successive  free-cell  formations  in  the  asco- 
gonium seems  highly  improbable,  though  Kuyper  agrees  with  him  on 
this  point.  Neither  Ikeno's  nor  Kuyper's  figures  are,  however,  con- 
vincing, and  to  establish  the  existence  of  a  process  of  forming  asci  or 
spore  mother  cells,  as  well  as  ascospores,  by  free  cell- formation  cer- 
tainly demands  the  presentation  of  a  detailed  account  with  good  figures 
of  the  stages  involved.  If  the  asci  or  "spore  mother  cells"  of  Monascus 
are  formed  by  free  cell-formation,  it  is  certainly  evidence  against  its 
close  relationship  with  the  Ascomycetes.  Kuyper  believes  it  should  be 
made  the  representative  of  a  new  family,  the  Endascineae,  connecting 
the  Ascomycetes  to  the  Oomycetes.  Ikeno  probably  calls  these  bodies 
"  spore  mother  cells  "  on  an  assumed  analogy  between  them  and  the 
cells  formed  by  free  cell-formation  in  Taphridium,  to  which  Juel  (51) 
has  given  this  name.  It  is  to  be  remembered,  however,  that  these  "  spore 
mother  cells  "  of  Taphridium  form  spores  not  by  free  cell-formation, 
but  by  ordinary  division.  It  is  hard  to  see  how  Ikeno's  account  of 
spore  formation  gives  any  basis  for  his  conclusion  that  his  Monascus 
has  relations  with  the  other  so-called  Hemiasci.  Kuyper  indulges  in 
some  very  sharp  criticism  of  the  lack  of  homogeneity  of  this  group, 
which  is  doubtless  justified,  though  we  are  more  in  need  of  new  facts 
about  the  forms  in  question  than  of  a  priori  criticism.  Still  later  papers 
by  Barker  (4)  reaffirm  the  correctness  of  his  account  of  the  reproduc- 
tion of  Monascus  and  add  a  preliminary  account  of  sexual  cell  fusion 
in  the  development  of  the  ascocarp  of  Rhyparobius.  In  this  form  we 
find  a  fusion  of  gametes  with  several  nuclei.  The  ascogonium  develops 


MORPHOLOGY    OF    THE    ASCOCARP.  27 

as  a  several-celled  structure  from  which  the  ascogenous  hyphae  arise. 
We  have  thus  a  fusion  of  gamete  cells  in  another  typical  discomycete. 

Baur  (5)  has  added  a  further  valuable  contribution  to  his  studies 
on  the  development  of  the  apothecia  of  the  lichens,  in  which  he  estab- 
lishes the  existence  of  carpogonia  and  trichogynes  in  a  further  series  of 
forms.  Especially  interesting  is  the  demonstration  of  carpogonia  and 
trichogynes  on  the  margins  of  the  podetia  of  Cladonia,  which  corrects 
the  mistaken,  though  generally  accepted,  conclusion  of  Krabbe  that  the 
entire  podetium  is  homologous  with  an  apothecium.  Baur  also  shows 
that  Wahlberg's  recent  conclusion  that  in  Anaptychia  the  paraphyses 
arise  from  the  ascogenous  hyphje  is  incorrect.  The  hyphse  which  sprout 
from  the  carpogonium  never  form  paraphyses.  Baur  also  adds  another 
form,  Solorina,  to  the  list  of  lichens  which  seem  plainly  apogamous. 
The  lichens  bid  fair  soon  to  become,  if  they  are  not  already,  the  best- 
known  group  of  the  higher  fungi  as  to  the  actual  facts  in  the  develop- 
ment of  their  fruiting  organs. 

Claussen  (16)  has  also  made  a  most  interesting  and  important  con- 
tribution to  our  knowledge  of  the  ascocarp  as  a  result  of  very  carefully 
conducted  studies  on  a  new  species  of  Boudiera.  By  growing  the  fungus 
in  appropriate  cultures  he  was  able  to  follow  the  development  of  the 
sexual  apparatus  and  the  fusion  of  the  gametes  in  living  material.  The 
apothecium  of  Boudiera  takes  its  origin,  like  that  of  Pyronema,  from 
several  pairs  of  gametes.  The  antheridium  and  oogonium  are  spirally 
coiled  together,  and  a  trichogyne  cell  is  cut  off  from  the  apex  of  the 
latter.  As  in  Pyronema,  the  fusion-pore  between  the  antheridium  and 
trichogyne  is  more  permanent,  while  the  disappearance  of  the  wall 
between  the  trichogyne  and  oogonium  is  transient  and  much  more  diffi- 
cult of  observation.  Claussen  did  not  find  this  opening  in  sections,  but 
was  able  to  determine  the  transitory  disappearance  of  the  wall  between 
the  trichogyne  and  oogonium  in  living  material.  The  ascogenous  hyphse 
arising  from  the  ascogonium  seem  each  to  produce  but  one  ascus  which 
is  typical  in  all  respects  in  its  development.  We  have  thus  a  further 
case  of  an  ascocarp  which  arises  from  a  compounded  sexual  apparatus. 
As  Claussen  points  out,  such  discoveries  as  these  will  furnish  data  for 
a  much  more  satisfactory  arrangement  of  the  groups  of  the  Ascomy- 
cetes  than  is  yet  possible. 

Juel  (52)  has  given  a  most  interesting  account  of  the  nuclear  phe- 
nomena in  the  fertilization  of  Dipodascus.  The  gametes  are  multinu- 
cleated,  but  only  a  single  pair  of  nuclei  fuse.  By  division  of  the  fusion- 
nucleus  the  spore-nuclei  are  formed.  In  view  of  Dangeard's  attempt 
to  homologize  each  ascus  of  the  higher  Ascomycetes  with  a  supposedly 


28  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

apogamous  fructification  of  Dipodascus,  it  is  interesting  to  note  that 
Juel  insists  that  the  spore-sac  of  Dipodascus  corresponds  to  the  entire 
cell  complex  which  arises  from  a  fertilized  carpogonium.  The  nuclear 
fusion  in  Dipodascus  corresponds  to  the  sexual  fusions  in  the  carpogo- 
nium and  not  to  those  in  the  asci.  This  is  the  view  adopted  by  Wager 
(97),  and  in  his  more  recent  discussions  Dangeard  (21)  has  given  up 
his  earlier  position,  since  plainly  the  individual  asci  in  his  fig.  5,  p.  55, 
do  not  correspond  to  the  fructifications  shown  in  fig.  4,  p.  154.  With 
this  the  contention  that  the  ascus  is  an  egg  becomes  still  more  incon- 
sistent. In  adopting  this  latest  conception  that  the  ascogonium  and 
ascogenous  hyphse  constitute  a  gametophore  derived  by  growth  and 
branching  from  a  gametangium  which  has  ceased  to  produce  motile 
gametes  in  adaptation  to  a  terrestrial  habit,  Dangeard  admits  De  Bary's 
contention  that  the  ascocarp  is  a  unit  morphologically  comparable  in  its 
initial  stages  to  one  or  more  pairs  of  oogonia  and  antheridia.  He  thus, 
in  reality,  rejects  his  earlier  conception  that  each  ascus  is  an  egg  and 
morphologically  the  equivalent  of  a  hypothetical  parthenogenetic  fruit- 
body  of  the  lower  fungi  or  algse.  Dangeard  now  inclines  to  the  view 
that  the  ancestry  of  the  Ascomycetes  is  to  be  sought  in  the  Oomycetes ; 
and  the  real  conclusion  from  his  argument,  admitting  his  premises,  is 
that  the  ascogonium  and  ascogenous  hyphse  are  vegetative  outgrowths 
from  a  parthenogenetic  egg  and  that  the  ascus  is  a  new  structure  in 
which  this  vegetative  development  ends. 

Dangeard  (21)  maintains  that  he  can  not  find  the  stage  when  fusion 
takes  place  in  Pyronema,  but  negative  evidence  on  a  point  of  this  kind, 
when  unaccompanied  by  positive  results  as  to  further  details  of  cell  and 
nuclear  structure  and  behavior,  are  of  little  value.  Meanwhile,  we  may 
be  sure  that  morphologically,  in  its  relation  to  the  fruiting  bodies  in 
other  groups  of  fungi  or  algse,  the  ascocarp  is  to  be  interpreted  as  origi- 
nating from  a  sexual  apparatus.  This  fact  will  not  be  altered  by  the 
discovery  of  forms  in  which,  by  apogamy  or  parthenogenesis,  the  sexual 
cells  have  either  been  modified  in  form  or  become  functionless. 

I  am  unable  to  find  anything  in  the  discussions  of  Dangeard  to 
invalidate  the  conclusions  which  I  reached  in  a  former  paper  (40)  as 
to  the  morphology  of  the  ascocarp.  Further,  the  positive  results  briefly 
summarized  above — obtained,  as  they  are,  by  different  workers  and  on 
widely  separated  forms — certainly  favor  the  conclusion  that  the  sexual 
organs  of  the  Ascomycetes  are  regularly  functional ;  and  when  we  con- 
sider the  difficulties  involved  in  actually  working  out  every  stage  in  the 
development  of  an  ascocarp  from  its  earliest  inception,  it  is  not  sur- 
prising that  our  knowledge  has  not  advanced  more  rapidly. 


MORPHOLOGY    OF    THE    ASCOCARP.  29 

Dangeard  persists  in  professing  himself  quite  indifferent  to  the 
existence  of  antheridia  and  oogonia  as  the  initial  cells  of  the  ascocarp, 
if  only  they  be  not  functional  at  the  present  time;  but  one  can  hardly 
believe  him  so  lacking  in  penetration  as  not  to  realize  that  so  long  as 
the  Ascomycetes  are  regarded  as  a  monophyletic  group  the  establish- 
ment of  the  existence  of  antheridia  and  oogonia  as  the  initial  cells  of 
the  ascocarp  settles  at  once,  and  finally,  the  old  question  of  the  sex- 
uality of  the  group  in  favor  of  the  views  of  De  Bary  and  his  school 
and  against  those  of  Brefeld — and  this,  too,  without  any  regard  to  the 
question  as  to  whether  these  sex  cells  are  functional  or  not.  Interesting 
evidence  on  this  point  is  shown  in  the  attitude  of  the  present  adherents 
of  the  views  of  Brefeld  (57),  who  are  solely  interested  in  the  question 
whether  archicarps,  pollinodia,  etc.,  are  really  sexual  cells,  and  appear 
relatively  indifferent  as  to  the  existence  of  the  fusions  in  the  asci.  No 
one  can  reasonably  doubt  that  the  antheridia  of  the  Saprolegniaceae  are 
sexual  germ-cells,  regardless  of  the  question  as  to  whether  they  are 
functional  at  the  present  time.  The  proof  brought  by  Trow  (95)  that 
a  fusion  of  cells  and  nuclei  occurs  at  the  present  time  is  confirmatory 
evidence  of  their  sexual  nature,  but  it  is  in  no  way  necessary  for  their 
correct  morphological  characterization  as  compared  with  corresponding 
structures  in  other  groups  of  algae  and  fungi.  It  was  the  supposed 
similarity  of  De  Bary's  antheridial  branch  in  the  mildews  to  the  later- 
developed  perithecial  branches  which  was  a  justification  for  doubt  as 
to  its  morphological  nature  and  made  it  necessary  to  show  that  it  was 
a  functional  male  branch  in  order  to  establish  its  morphological  char- 
acter. Could  De  Bary  have  brought  such  evidence  of  the  special  differ- 
entiation of  its  walls  and  its  fate  in  the  developing  perithecium  as  can 
be  found  by  modern  methods  in  Phyllactinia,  in  addition  to  the  facts  as 
to  its  origin,  position,  etc.,  we  can  hardly  imagine  that  its  sexual  nature 
would  ever  have  been  doubted,  regardless  of  whether  it  was  shown  to 
be  functional  at  the  present  time.  The  ascus  is  in  its  origin  a  new 
structure,  the  outgrowth  of  ascogenous  hyphse  and  ascogonium,  and 
the  fusion  of  nuclei  which  occurs  in  it,  whatever  may  be  its  physiological 
nature,  is  not  homologous  with  the  fertilization  of  the  egg  out  of  which 
the  ascogonium  develops.  It  is  thus  fairly  established  and  admitted 
that  the  "  macrocysts  and  paracysts  "  of  Pyronema  and  the  "  archicarp 
and  pollinodium  "  of  the  Erysipheae  and  other  Ascomycetes  are  to  be 
compared  morphologically  to  the  gametangia  and  oogonia  and  anther- 
idia of  other  fungi  and  algae,  and  that  the  ascogonium,  ascogenous 
hyphae,  and  asci  are  new  structures  gradually  developed  in  the  evolu- 
tion of  reproductive  processes,  just  as  are  the  gonimoblasts  and  carpo- 
spores  of  the  red  algae. 


3O  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

SPECIAL   NUCLEAR    PHENOMENA. 

Phyllactinia  is  especially  favorable  for  the  examination  of  the 
structure  of  the  nuclei  and  their  fusion  and  division  in  the  ascogenous 
hyphse  and  asci.  The  large  number  of  asci  formed  in  each  ascocarp 
makes  it  possible  to  readily  obtain  large  numbers  of  the  nuclei  in  all 
stages  of  their  development.  The  young  perithecia  of  Phyllactinia 
also,  in  most  of  their  stages,  seem  especially  favorable  for  fixation,  so 
that  the  nuclear  figures  in  the  sexual  cells,  the  ascogenous  hyphse,  and 
young  asci  are  differentiated  with  exceptional  distinctness  and  clearness 
of  detail.  As  a  result,  certain  structures  which  could  be  made  out 
only  at  particular  stages  and  without  full  details  in  Erysiphe  can  be 
found  at  every  stage  of  nuclear  development  in  Phyllactinia.  I  have 
described  (38)  for  Erysiphe  a  peculiar  and  characteristic  attachment  of 
the  chromatin  of  the  nucleus  to  the  central  body,  giving  the  nucleus  a 
characteristic  polar  rather  than  a  radial  structure.  This  condition  is 
very  conspicuous  in  the  young  daughter  nuclei  in  the  ascus  of  Erysiphe. 
In  Phyllactinia  the  nuclei  throughout  the  entire  plant,  in  both  the 
mycelium  and  ascocarps,  show  this  characteristic  relationship  of  the 
chromatin  and  the  central  body;  and  in  the  ascogenous  hyphse  and 
young  asci  a  very  definite  type  of  orientation  of  the  chromatin  threads 
as  a  cone  or  partial  aster  extending  from  the  central  body  into  the  cavity 
of  the  nucleus  is  conspicuous.  This  persists  through  the  fusion  stages 
in  the  ascus  as  well  as  through  division  and  the  resting-stages,  and  the 
central  body  is  thus  shown  to  be  a  permanent  structure  through  the 
whole  life  history  of  the  mildews. 

I  shall  continue,  as  in  former  papers,  to  call  this  structure  a  central 
body  rather  than  to  use  the  term  centrosphere  or  centrosome.  I  do  this 
not  to  indicate  that  I  consider  this  body  in  the  mildew  as  different  from 
apparently  similar  structures  found  in  the  karyokinetic  figures  of  other 
plants,  but  merely  because  I  prefer  the  more  general  descriptive  term 
rather  than  a  more  technical  one,  since  the  bodies  in  question  are  still  so 
variously  described  by  the  different  authors  who  have  worked  on  them. 

It  is  a  conspicuous  and  important  fact  that  the  nuclei  and  cells  of 
the  mildews  undergo  extreme  variation  in  size  in  the  course  of  the 
development  of  the  fungus,  and  in  general  it  is  plain  that  the  nuclei  are 
larger  in  the  larger  cells.  Before  proceeding  with  the  description  of 
the  organization  of  the  nucleus  I  shall  briefly  summarize  the  facts  as 
to  this  variation,  since,  as  we  shall  see  later,  these  facts  may  have  an 
important  bearing  on  the  interpretation  of  the  nuclear  fusion  in  the  ascus. 
The  nucleus  of  the  oogonium  is  considerably  larger  (fig.  7)  than  the 


SPECIAL    NUCLEAR    PHENOMENA.  3! 

ordinary  hyphal  nuclei.  The  nuclei  of  the  stalk  cells  of  both  oogonium 
and  antheridium,  from  which  the  protective  hyphse  are  to  sprout,  are 
frequently  larger  (figs.  7,  10),  at  least  at  the  stages  when  the  formation 
of  the  perithecium  is  about  to  begin,  than  those  of  the  ordinary  hyphal 
cells,  though  not  as  large  as  the  nucleus  of  the  oogonium.  As  in  many 
other  cases,  the  male  nucleus  is  regularly  smaller  than  that  of  the  egg, 
just  as  the  antheridium  is  smaller  than  the  oogonium  (figs.  5,  6,  7,  8,  9). 

After  fertilization,  with  the  growth  of  the  ascogonium  its  nuclei 
become  still  larger,  and  in  the  young  ascus  the  pair  of  nuclei  which  fuse 
are  conspicuously  larger  than  any  that  have  preceded  them  (figs.  31-33), 
and  the  product  of  their  union,  the  primary  nucleus  of  the  ascus,  is  one 
of  the  largest  nuclei  to  be  found  among  the  fungi  (figs.  48,  49).  It  is 
much  larger  than  the  nuclei  of  the  cells  of  the  leaf  on  which  the  mildew 
grows.  Its  diameter  is  about  10  /*  as  compared  with  a  diameter  of  2  /u, 
in  the  hyphal  nuclei.  This  large  size  of  the  primary  nucleus  of  the 
ascus  may  well  be  regarded  as  correlated  with  the  large  size  of  the  ascus 
itself.  Roughly,  the  volume  of  the  nucleus  of  the  ascus  seems  to  stand 
in  a  similar  proportion  to  the  volume  of  the  entire  ascus  as  the  volume 
of  a  hyphal  nucleus  does  to  the  cell  which  contains  it.  The  cells  of  the 
full-grown  perithecium  contain  several  nuclei  which  are  rather  smaller 
than  those  of  the  mycelial  cells,  which  are  regularly  uninucleated. 
When  the  primary  nucleus  of  the  ascus  divides,  the  daughter  nuclei  are 
certainly  not  more  than  half  as  large  (fig.  62)  as  the  parent  nucleus. 
The  size  of  the  nuclei  produced  in  the  second  and  third  divisions  is  also 
proportionally  reduced,  and  finally  the  nuclei  of  the  ascospores  are  of 
about  the  size  of  the  ordinary  nuclei  of  the  young  perithecium  (fig.  79). 
There  can  be  no  doubt  that  we  have  here  a  definite  correlation  between 
nuclear  and  cytoplasmic  masses,  such  that  the  larger  cells  contain 
proportionally  larger  nuclei,  and  we  have  thus  an  illustration  of  the 
principle  of  the  nucleo-cytoplasmic  relation  developed  by  R.  Hertwig, 
Gerassimoff,  and  Boveri. 

The  nucleus  of  the  oogonium  is,  as  noted,  considerably  larger  than 
those  of  the  vegetative  hyphse.  The  oogonial  cytoplasm  is  quite  dense 
and  shows  a  fine,  close,  spongy  structure  (figs.  1-7).  The  central  body 
of  the  oogonial  nucleus  is  a  conspicuous,  well-differentiated,  disk-shapecl 
granule  (figs.  4,  5,  7)  lying  close  on  the  surface  of  the  nuclear  mem- 
brane and  generally  occupying  a  slight  depression  in  it.  The  arrange- 
ment of  the  chromatin  content  of  the  nucleus  can  not  be  so  clearly  made 
out  at  this  stage  as  at  later  stages  in  the  larger  nuclei  of  the  ascogenous 
hyphse  and  the  asci ;  still  it  is  perfectly  plain,  in  every  case  in  which  the 
plane  of  the  section  permits  a  profile  view  of  the  structure,  that  the 


32  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

chromatin  is  in  intimate  connection  with  the  central  body  (figs.  I,  3,  4, 
5,  7),  and  in  specially  favorable  preparations  (figs.  4,  7)  it  is  clear  that 
there  are  strands  in  the  chromatin  reticulum  which  radiate  back  from 
the  central  body  into  the  cavity  of  the  nucleus,  forming  an  intranuclear 
system  almost  like  an  aster  whose  rays  appear  quite  straight  and  definite 
in  many  cases,  though  they  are  connected  by  transverse  fibrilbe  and  lose 
themselves  in  a  more  indefinite  granular  reticulum  in  the  region  oppo- 
site the  central  body.  The  nucleus  has  plainly,  at  this  stage,  a  unipolar 
structure,  and  the  chromatin  is  definitely  oriented  with  reference  to  the 
central  body.  Following  Rabl's  terminology,  I  shall  speak  of  that  part 
of  the  nucleus  which  is  antipodal  to  the  central  body  as  the  antipolar 
region  and  the  region  about  the  central  body  as  the  polar  region. 

The  nucleus  of  the  oogonium  has  regularly  a  single  oval  or  spher- 
ical dense  homogeneous  nucleole  (figs.  1-7),  which  commonly  lies  some- 
where in  the  antipolar  region,  but  may  frequently  lie  close  to  the  central 
body,  crowding  aside  the  chromatin  elements. 

The  nucleus  of  the  antheridial  cell  is  still  smaller  prior  to  fusion 
than  that  of  the  oogonium ;  still  its  central  body  is  sharply  differentiated 
and  the  chromatin  is  plainly  connected  with  it  (figs.  5,  6).  It  has  also 
regularly  a  single  nucleole.  The  structure  of  the  nucleus  of  the  anther- 
idial stalk-cell  and  of  the  other  vegetative  nuclei  agrees  with  that  of  the 
sexual  cells  (figs.  4,  5,  7).  Frequently  the  hyphal  nuclei  are  oval  or 
more  elongated,  and  may  be  drawn  out  to  a  point  on  which  the  central 
body  sometimes  lies.  (Compare  figs.  24  and  14).  After  its  migration 
into  the  oogonium  the  antheridial  nucleus  enlarges  and  the  two  pro- 
nuclei  are  each  plainly  seen  to  be  provided  with  central  bodies  at  a  stage 
just  before  they  fuse  (fig.  9).  The  fertilized  egg-nucleus  also  shows 
a  single  conspicuous  center  (figs.  10,  II,  12),  and  it  seems  probable  that 
it  has  been  formed  by  the  combination  of  the  centers  of  the  pronuclei. 
It  is  also  probable  that  this  central  body  of  the  fertilized  egg- 
nucleus  is  actually  double  as  compared  with  those  of  the  pronuclei,  since, 
as  we  shall  see  later,  we  have  strong  evidence  of  the  permanence  of  the 
chromosomes  as  cell  structures,  and  a  nuclear  fusion  should  hence  pro- 
duce a  nucleus  with  a  double  number  of  chromosomes,  each  of  which 
would  have  an  independent  attachment  to  the  central  body.  But  I  have 
not  been  able  to  obtain  a  sufficient  series  of  preparations  at  this  stage 
to  be  able  to  trace  the  process  of  the  combination  of  the  chromatin  and 
centers  in  this  fusion,  nor  to  determine  the  number  of  chromosomes  in 
the  next  succeeding  division.  The  fertilized  egg-nucleus  regularly  con- 
tains a  single,  rather  large  nucleole  (figs.  10, 12),  which,  it  seems  prob- 
able, is  also  formed  by  the  fusion  of  the  nucleoles  of  the  pronuclei. 


SPECIAL    NUCLEAR    PHENOMENA.  33 

With  the  growth  of  the  ascogonium  the  egg-nucleus  divides  and  the 
daughter  nuclei  become  successively  larger  with  the  increasing  size  of 
the  cells  in  which  they  lie.  This  growth  continues  till  the  ascogonium 
and  ascogenous  hyphse  reach  their  full  development,  as  described  above. 

The  nuclei  appear  at  this  stage  in  pairs  in  all  the  cells  of  the  asco- 
genous hyphas  which  are  destined  to  produce  asci  (figs.  28,  29,  31-33). 
The  question  as  to  the  probable  relationship  of  these  nuclei  has  been 
discussed  above.  There  is  no  such  arrangement  for  preventing  the 
inclusion  of  sister  nuclei  in  the  ascus  as  I  have  described  for  Pyronema 
(40).  On  the  other  hand,  the  ascogenous  hyphse  are  multinucleated 
before  cell  division  occurs,  and  there  is  no  direct  evidence  that  the 
nuclear  pairs  are  formed  by  the  division  of  a  single  nucleus. 

The  structure  of  these  larger  nuclei  (figs.  31-33)  appears  to  be  the 
same  as  that  of  the  sexual  nuclei  just  described,  but  owing  to  their 
greater  size  the  details  can  be  made  out  with  greater  definiteness.  The 
relations  of  the  central  body  and  nuclear  content  come  out  much  more 
sharply,  and  it  is  possible  to  count  with  some  certainty  the  number  of 
chromatin  strands  which  extend  into  the  nuclear  cavity  from  the  central 
body.  The  nucleole  stains  bright  red  with  safranin  and  is  frequently 
flattened  somewhat  against  the  nuclear  membrane,  even  tending,  at 
least  in  fixed  material,  to  break  through  it  (figs.  31,  33).  The  flattened 
disk-like  form  of  the  central  body,  as  it  lies  pressed  against  the  nuclear 
membrane,  can  be  clearly  made  out.  Its  appearance  and  staining  reactions 
are  the  same  as  I  have  already  described  (38)  for  the  central  bodies  of 
other  mildews  and  Discomycetes.  With  the  triple  stain  the  chromatin 
shows  a  bright-blue  color  and  the  central  body  is  reddish  or  violet. 

The  strands  of  chromatin  are  regularly  attached  to  the  central 
body,  and  from  this  point  they  extend  into  the  central  cavity  of  the 
nucleus,  forming  a  sheaf  of  diverging  rays.  The  series  of  threads  as  a 
whole  produces  distinctly  the  effect  of  a  cone  or  bundle  of  rays  extend- 
ing from  the  central  body  into  the  cavity  of  the  nucleus  (figs.  31-34). 
The  bundle  is  broader  or  narrower,  according  as  the  threads  diverge 
more  or  less  rapidly  from  their  point  of  attachment.  In  some  cases  the 
outer  rays  may  follow  more  or  less  closely  for  some  distance  the  inner 
surface  of  the  nuclear  membrane,  and  the  whole  system  of  threads  may 
thus  be  distributed  quite  evenly  through  the  nuclear  cavity.  In  this 
case  the  appearance  of  a  cone  or  bundle  of  threads  diverging  from  the 
central  body  is  partially  lost,  but  the  orientation  and  attachment  of 
the  threads  on  the  center  is  distinct  and  definite.  In  the  majority  of 
cases,  however,  in  this  stage  the  threads  form,  for  some  distance  inward 
from  the  center,  quite  a  definite  diverging  bundle  (figs.  33,  34).  If  we 


34  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

endeavor  to  follow  the  paths  of  the  individual  threads  we  find  them 
either  straight  or  wavy  in  outline,  and  not  infrequently  threads  are  found 
which  bend  sharply  this  way  and  that,  following  no  definite  direction. 
Such  threads  are,  however,  relatively  few  and  never  abundant  enough 
to  interfere  with  the  general  appearance  of  a  radiating  system  centered 
on  the  central  body.  Fig.  34  shows  a  section  cut  from  one  side  of  a 
nucleus,  so  as  to  include  only  three  chromatin  strands  with  fragments 
and  cross-sections  of  others.  In  composition  the  threads  appear  lumpy 
or  finely  granular,  and  hence  of  quite  irregular  thickness  and  density, 
since  the  nodules  are  by  no  means  of  equal  size.  They  probably  consist 
of  chromatin  granules  embedded  in  and  connected  by  a  less  stainable 
linin,  though  the  distinction  of  chromatin  and  linin  can  scarcely  be  made 
out  at  this  stage.  The  main  threads  do  not  apparently  anastomose, 
though  they  are  occasionally  found  crossing  each  other.  They  are, 
however,  connected  by  lateral  fibrillar  or  granular  extensions  which  are 
extremely  delicate  and  quite  numerous,  so  that  in  sections  showing  the 
entire  nucleus  (fig.  32)  the  more  prominent  threads  appear  almost  as  if 
embedded  in  a  very  faintly  blue-stained  ground  substance.  In  fig.  34 
the  antipolar  ends  of  the  strands  seem  very  definite,  but  this  is  probably 
due  to  the  fact  that  they  are  cut  off  in  sectioning.  In  figures  showing 
the  entire  nucleus  (figs.  32-34),  and  especially  in  the  smaller  nuclei  of 
a  slightly  earlier  stage  (fig.  31),  it  is  plain  that  the  farther  one  follows 
the  threads  from  the  central  body  across  the  diameter  of  the  nucleus  the 
more  difficult  it  is  to  distinguish  them.  In  the  nuclei  prior  to  their 
fusion  it  is  impossible  to  trace  the  threads  across  the  entire  diameter  of 
the  nucleus.  They  seem  to  fade  out  and  lose  their  identity  in  a  less 
strongly  differentiated  granular  and  thready  mass,  whose  structure  and 
relation  to  the  main  chromatin  strands  is  not  easy  to  make  out  clearly. 
The  whole  system  of  threads  seems  to  pass  over  very  soon  (in  fig.  31) 
into  a  less  differentiated  granular  reticulum  composed  of  deeply  stained 
chromatin  granules  connected  with  abundant,  more  faintly  stained 
fibrillae,  which  seem  almost  to  form  a  ground  substance.  The  fibrillae 
resemble  closely  the  faintly  stained  fibrous  material  which  connects 
the  threads  nearer  the  center.  This  condition  in  the  antipolar  portion 
of  the  nucleus  doubtless  represents  more  nearly  a  resting  condition  of 
the  nuclear  material  in  which  the  chromatin  is  more  irregularly  scat- 
tered in  the  nuclear  cavity,  but  is  still  connected  definitely  with  the 
central  body. 

The  appearance  of  the  chromatin  in  the  resting  nuclei  in  the  myce- 
lial  hyphae  and  the  cells  of  the  perithecium  (figs.  23,  24)  is  very  similar 
to  that  which  we  find  in  the  antipolar  region  of  these  nuclei  of  the 


SPECIAL    NUCLEAR    PHENOMENA.  35 

young  ascus.  I  have  elsewhere  described  (37)  such  mycelial  nuclei 
as  finely  granular,  and  the  description  applies  to  the  vegetative  nuclei 
of  Phyllactinia.  It  is  clear  that  these  granules  are  the  chromatin 
elements  in  a  finely  divided  and  distributed  condition,  such  as  has  been 
commonly  associated  with  the  resting  condition  of  the  nucleus.  In 
all  the  vegetative  resting  nuclei  of  Phyllactinia,  however,  the  central 
body  is  in  intimate  connection  by  delicate  fibrillse  with  this  granular 
content  of  the  nucleus,  and,  since  the  antipolar  region  of  the  larger 
nuclei  of  the  young  asci  shows  a  similar  granular  appearance  with  the 
linin  fibrillse  connecting  the  granules  into  a  reticulum,  it  is  probable  that 
this  represents  the  structure  of  the  resting  nuclei  generally  in  this 
mildew.  In  the  resting  nucleus  the  chromatin  threads  are  so  loosely 
distributed  and  are  connected  so  frequently  by  linin  fibers  as  not  to  be 
clearly  distinguishable,  the  appearance  being  that  of  a  reticulum;  but 
the  attachment  of  the  chromatin  threads  to  the  central  body  is  contin- 
uous and  becomes  conspicuous  in  the  process  of  aggregation  by  which 
the  apparently  scattered  granules  of  the  nuclear  reticulum  of  the  resting 
stage  are  transformed  into  the  spirem  thread. 

Tracing  the  course  of  any  particular  chromatin  thread,  then,  at 
this  stage,  we  may  say  that  it  is  attached  at  one  end  on  the  central  body, 
passes  back  through  the  nucleus  in  a  path  which  may  be  rigidly  straight 
or  more  or  less  wavy  or  bent,  and  either  ends  freely  or  seems  to  fade  out 
in  the  apparently  less  differentiated  granular  reticulum  of  the  antipolar 
region.  The  whole  series  of  threads  forms  a  broader  or  narrower  cone 
or  pencil  of  coarse  rays  extending  from  the  central  body  into  the  nucleus 
and  filling  more  or  less  completely  the  nuclear  cavity.  The  outline  of 
the  more  dense  portions  of  the  nucleus  is  determined  by  the  outline  of 
the  chromatin  system.  The  nuclear  membrane  may  lie  on  the  surface 
of  this  chromatin  mass  or  it  may  be  separated  from  it  by  a  zone  of  clear 
non-stainable  nuclear  sap  (fig.  31).  Frequently  this  zone  of  nuclear 
sap  extends  around  the  whole  surface  of  the  chromatin  system,  except 
where  the  threads  are  fastened  to  the  central  body.  The  relations  of 
the  threads  to  the  centers  is  much  emphasized  in  such  cases,  in  that  the 
center  is  the  only  point  in  which  the  chromatin  is  in  contact  with  the 
nuclear  membrane,  and  thus  with  the  cytoplasm  of  the  cell.  Such 
figures  indicate,  as  I  have  suggested  in  an  earlier  paper  (38),  that  the 
central  body  represents  a  special  region  of  connection  between  the 
interior  of  the  nucleus  and  the  cytoplasm. 

The  connection  of  the  nuclear  threads  with  the  central  body  implies, 
of  course,  that  the  nuclear  membrane  is  not  continuous  at  this  point,  or 
at  least  that  it  permits  in  some  way  the  connection  of  the  threads  with 


36  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

the  pole.  In  the  prophases  of  the  division  of  the  nuclei  in  the  asci  of 
£.  communis  the  connection  frequently  appears  partially  ruptured  (38, 
Taf.  n,  figs.  4,  n).  This  may  be  due  to  the  fixation.  Occasionally 
the  undifferentiated  portion  of  the  chromatin  reticulum  rests  also  on 
the  nuclear  membrane  over  the  whole  antipolar  region,  and  this  may 
possibly  be  regarded  as  the  more  normal  condition.  The  nuclear  mem- 
brane is  spherical  in  such  cases,  and  the  only  region  of  clear  nuclear 
sap  is  that  left  by  the  drawing  together  into  a  cone  of  the  chromatin 
threads  which  are  converging  on  the  central  body. 

In  some  cases  the  zone  of  clear  nuclear  sap  extending  around  the 
entire  chromatin  system,  except  at  the  pole,  is  so  wide  as  to  suggest 
that  the  nuclear  membrane  has  swelled  away  from  the  nuclear  contents 
in  fixation.  Such  figures,  assuming  that  they  indicate  swelling  of  the 
nuclear  membrane,  give  evidence  of  the  firmness  of  the  attachment  of 
the  chromatin  threads  to  the  central  body  and  of  the  latter  to  the  cyto- 
plasm, since  in  well-fixed  material  the  chromatin  is  never  separated 
from  the  center  nor  the  latter  from  the  cytoplasm,  no  matter  how  wide 
the  clear  zone  may  be  about  the  remainder  of  the  chromatin  system. 

Sometimes  also  a  clear  zone  is  formed  about  the  nucleus  outside 
the  nuclear  membrane,  such  as  has  been  described  for  the  nuclei  of 
Chara;  but  in  this  case  the  central  body  is  neither  separated  from  the 
nuclear  membrane  nor  the  cytoplasm,  indicating  again  its  connection 
with  both  of  those  structures.  Not  infrequently  the  chromatin  threads 
at  this  stage  show  parallel  bends  and  curves,  and  I  find  many  figures  in 
which  the  whole  system  tends  to  be  slightly  spirally  twisted.  There  is 
also  some  evidence  that  the  threads  are  arranged  in  pairs. 

When  the  young  ascus  has  reached  the  stage  shown  in  fig.  33,  the 
pair  of  nuclei  fuse  to  form  the  primary  nucleus  of  the  ascus.  The 
process  of  fusion  can  be  followed  in  all  its  details  with  relatively  great 
readiness  in  Phyllactinia,  owing  to  the  large  number  of  asci  in  each 
perithecium.  At  the  time  of  fusion  the  ascus-cell  consists  of  an  upper 
enlarged  portion,  in  which  the  nuclei  lie,  and  a  lower  stalk-like  portion, 
which  is  much  narrower  and  extremely  irregular  in  its  shape,  twisting 
about  among  the  wall-cells  of  the  base  of  the  perithecium  and  connecting 
with  the  original  system  of  the  ascogenous  hyphae  from  which  it  devel- 
oped. A  section  of  the  upper  portion  of  the  ascus  rarely  shows  this 
elongated  narrower  portion  in  its  entire  length,  since  the  two  rarely  lie 
in  the  same  vertical  plane.  Later,  with  the  growth  of  the  perithecium, 
this  stalk-like  portion  of  the  ascus  swells  and  becomes  a  part  of  the 
oblong  ascus-sac,  except  at  its  lowest  portion,  which  still  remains  as  a 
narrowed  foot  or  stalk. 


SPECIAL    NUCLEAR    PHENOMENA.  37 

The  cytoplasm  of  the  ascus  is  homogeneous  and  spongy,  with  no 
large  vacuoles  or  inclusions  at  this  stage.  The  nuclei  lie  rather  close 
together,  separated  by  a  less  distance  than  their  own  diameter  through- 
out the  early  development  of  the  ascus  (figs.  31,  32).  They  fuse  at  a 
stage  just  before  the  penicillate  cells  begin  to  sprout  out  on  the  upper 
surface  of  the  perithecium.  As  they  approach  each  other  to  fuse,  the 
central  bodies  may  be  very  variously  placed  with  reference  to  each  othei 
and  to  the  plane  of  contact  of  the  nuclei.  I  have  been  unable  to  find  any 
evidence  that  the  position  of  the  centers  influences  in  any  way  the 
approach  of  the  nuclei  or  determines  the  point  of  their  first  contact. 
The  centers  are  frequently  placed  facing  each  other  (fig.  31).  In 
other  cases  they  are  separated  by  90  or  more  degrees  on  the  surface  of 
the  nuclei  (fig.  33).  Occasionally,  just  prior  to  fusion,  one  nucleus 
may  be  pulled  out  into  a  pear-shaped  body,  with  the  center  at  its  nar- 
rowed end,  and  this  narrowed  end  may  be  extended  beside  the  second 
nucleus.  In  this  case,  however,  the  centers  may  still  be  separated  by 
half  a  circumference  from  each  other. 

The  nuclear  membrane  disappears  at  the  point  of  contact,  and  the 
chromatin  and  nucleoles  of  the  two  nuclei  thus  come  to  lie  in  a  common 
cavity.  In  fig.  35  one  chromatin  system  is  drawn  out  into  a  long  cone, 
the  central  body  apparently  having  pushed  ahead  into  the  second  nucleus, 
dragging  the  chromatin  after  it.  The  chromatin  threads  maintain  for 
some  time  their  independent  orientation  about  their  separate  central 
bodies  (figs.  35-37).  The  outline  of  the  double  nucleus  may  for  a  time 
show  a  constriction  in  the  plane  of  fusion ;  later  it  may  round  out  on  one 
side  before  it  does  on  the  other  (fig.  38) .  The  centers  are  still  variously 
placed  with  reference  to  each  other.  In  rare  cases  they  may  even  be 
for  a  time  exactly  opposite  each  other  on  the  surface  of  the  nucleus, 
with  their  respective  chromatic  systems  extending  toward  each  other, 
suggesting  the  formation  of  a  spindle.  Frequently,  however,  the  fusion 
occurs  in  such  a  way  that  the  centers  are  brought  close  together  at  once. 

Wherever  the  centers  may  be,  the  masses  of  chromatin  show  no 
tendency  at  this  stage  to  combine;  they  may  be  in  contact  with  each 
other,  but  are  simply  crowded  together  and  show  no  tendency  to  unite. 
Later,  in  all  cases  the  centers  are  found  lying  side  by  side  in  preparation 
for  fusing,  but  the  chromatin-thread  systems  are  still  quite  independent 
(fig.  38) .  At  this  stage,  as  at  the  stage  just  before  fusion,  it  is  possible 
to  count  approximately  the  number  of  threads  extending  back  from  each 
center.  There  are,  as  a  rule,  at  least  four  or  five  threads  lying  in  about 
the  same  focal  plane  near  the  median  optical  section  of  the  system.  By 
focusing  up  and  down  at  least  four  more  threads  can  be  made  out  which 


38  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

pass  up  or  down  from  the  center  so  as  not  to  lie  in  the  median  plane  of 
the  nucleus  as  a  whole.  There  are  then,  approximately  at  least,  eight 
or  nine  threads  in  each  system  just  before  fusion  occurs.  The  exact 
number,  of  course,  is  difficult  to  determine  in  every  case,  since  in  such 
minute  objects  it  is  difficult  to  be  sure  in  focusing  up  and  down  that 
different  threads  are  not  confounded  with  each  other  in  certain  portions 
of  their  extent,  or  that  some  threads  do  not  escape  notice  altogether 
by  being  hidden  behind  others  lying  in  nearly  the  same  vertical  plane. 

The  two  centers  can  be  found  closer  and  closer  together,  and  finally 
they  combine  side  by  side,  forming  at  first  a  double  center,  from  which 
the  distinct  sets  of  fibers  can  be  still  made  out  (fig.  38).  A  little  later 
the  two  sets  of  fibers  are  no  longer  distinguishable  (fig.  39).  The 
centers  at  the  time  of  fusion  maintain  their  ordinary  disk-like  shape, 
and  it  is  plain  that  the  fusion  figures  will  have  a  different  appearance 
according  as  the  centers  lie  side  by  side  or  one  above  the  other.  The 
former  are,  of  course,  much  more  favorable  for  study. 

The  nucleoles  also  fuse  at  some  time  during  the  process  of  the 
fusion  of  the  nuclei.  They  are  extremely  conspicuous,  bright-red  glob- 
ules, dense  and  homogeneous,  and  most  sharply  distinguishable  by  their 
red  color  in  the  triple  stain  from  the  blue-stained  chromatin.  Each 
nucleus  contains  without  exception  a  single  nucleole,  and  when  the 
nuclear  membranes  disappear  in  the  process  of  fusion  the  two  may  be 
very  variously  placed,  as  shown  in  the  figures.  Later  they  approach 
or  are  brought  together  in  the  movements  of  the  other  parts  of  the 
nucleus,  and  when  once  in  contact  they  remain  together  and  gradually 
fuse  into  a  single  nucleole  whose  diameter  is  conspicuously  greater  than 
that  of  either  of  the  two  which  fuse.  (Compare  figs.  31-33  with  36,  37, 
39,  42.)  If  they  had  a  perfectly  spherical  shape  it  would,  of  course, 
be  easy  to  determine  the  exact  relation  of  the  volumes  of  the  two  which 
fuse  with  that  of  the  resulting  nucleoles.  They  are,  however,  frequently 
somewhat  oblong  or  flattened  on  one  side,  and  since  it  is  not  easy  to 
determine  their  vertical  diameters  in  any  given  case,  exact  measurements 
of  their  volume  can  not  be  obtained.  As  is  seen  from  figs.  36-42,  the 
fusion  of  the  nucleoles  may  be  completed  either  before  or  after  the  fusion 
of  the  centers.  The  nucleoles  at  this  stage  always  lie  outside  the  chro- 
matin systems  and  their  fusion  seems  to  be  an  entirely  distinct  process. 

The  process  of  nuclear  fusion  may  be  summed  up  as  a  union  of  the 
two  resting  nuclei  into  a  single  spherical  nucleus  whose  volume  is  much 
greater  than  that  of  either  of  the  single  nuclei,  whether  or  not  it  is 
exactly  equal  to  their  sum.  The  nuclear  sap  of  the  two  makes  a  single 
homogeneous  non-stainable  liquid.  The  centers  fuse  into  a  single  larger 


SPECIAL    NUCLEAR    PHENOMENA.  39 

disk-shaped  center  and  the  chromatin  systems  have  become  intimately 
intermingled,  though  there  is  still  evidence  for  some  time  of  the  presence 
of  a  greater  number  of  threads  in  the  combined  chromatin  masses  than 
were  present  in  either  of  the  fusing  masses.  The  nucleoles  combine 
into  a  single  homogeneous  nucleole  of  approximately  twice  the  volume 
of  either  of  those  which  combined.  The  process  of  union  so  far  has 
gone  on  rather  rapidly;  at  least  there  is  no  evidence  of  increased  size 
in  the  entire  perithecium  between  the  stages  when  the  fusion  begins 
and  the  time  when  it  is  completed.  The  nucleus  thus  formed  is  the 
so-called  primary  nucleus  of  the  ascus. 

The  ascus  has  reached  about  half  its  mature  dimensions  at  this 
stage,  but  the  whole  perithecium  is  rather  more  than  half  grown.  As 
noted,  the  penicillate  cells  have  begun  to  develop,  but  the  characteristic 
bulbous-based,  spine-like  appendages  are  not  yet  present.  A  relatively 
long  period  now  ensues,  leading  up  to  the  spirem  stage  in  the  prophases 
of  the  first  division.  The  nucleus  during  this  period  grows  with  the 
growth  of  the  ascus  and  also  undergoes  characteristic  changes,  part  of 
which  constitute  essential  stages  of  the  fusion  process. 

In  the  nuclei  at  the  time  of  fusion,  and  immediately  thereafter,  the 
chromatin  substance  extends  through  a  large  part  of  the  nuclear  cavity 
(fig.  39),  as  was  described  above  for  the  stage  preceding  fusion.  The 
chromatin  threads  are  still  abundantly  connected  by  fibrillse,  and  these 
seem  to  become  more  numerous  and  delicate,  so  that  their  outlines  can 
scarcely  be  made  out  and  the  chromatin  strands  appear  as  if  they  were 
embedded  in  a  more  faintly  stained  ground  substance.  As  a  result  the 
threads  become  progressively  more  difficult  to  follow,  and  it  is  less  easy 
to  count  them.  The  whole  chromatin  mass  now  begins  to  contract  and 
becomes  more  dense  (fig.  43).  This  contraction  is  always  away  from 
the  antipolar  region  and  toward  the  center,  as  if  a  contraction  of  the 
threads  had  taken  place  by  which  they  are  drawn  up  to  their  points  of 
attachment  in  the  center.  The  antipolar  region  is  thus  left  almost  free 
of  stainable  materials  except  for  the  large  red-stained  nucleole.  With 
the  completion  of  this  stage  of  contraction  the  threads  may  become  more 
plainly  visible  (fig.  44),  but  they  are  much  shorter.  Frequently  the 
free  ends  of  the  threads  stick  out  from  the  denser  mass  and  are  plainly 
shown,  at  this  stage  at  least,  not  to  be  connected  as  loops  at  their  anti- 
polar  extremities  (fig.  44).  Occasionally  a  thread  may  extend  from 
the  mass  as  far  as  the  nucleole. 

There  is  evidence  in  some  cases  also  of  the  presence  of  a  consid- 
erable amount  of  faintly  stained  thready  material  which  may  appear  in 
section,  extending  from  the  surface  of  the  denser  mass  as  a  sort  of 


4O  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

fringe.  It  is  plainly  identical  with  the  fibrillar  material  of  the  earlier 
stages.  This  contracted  stage  lasts  for  some  time  if  estimated  in  terms 
of  the  growth  of  the  ascocarp. 

While  the  chromatin  is  thus  drawn  up  to  the  centers  the  outlines 
of  the  latter  are  difficult  to  make  out  in  preparations  stained  so  as  to 
give  the  chromatin  a  deep-blue  color.  The  whole  chromatin  mass  in 
deeply  stained  specimens  may  appear  as  a  roughly  oval  body  pressed 
against  the  nuclear  membrane  in  the  region  of  the  central  body.  If, 
however,  the  staining  be  modified  by  increasing  the  time  of  exposure 
to  the  orange  G  of  the  triple  stain,  until  the  blue  is  removed  from  the 
chromatin,  leaving  it  a  pale-gray,  the  center  appears  with  its  customary 
disk-like  shape  and  showing  a  dense  violet  color.  With  this  treatment 
the  red  nucleole  and  the  violet  center  are  the  only  deeply  stained  por- 
tions of  the  entire  ascus.  The  chromatin  is  pale  gray  and  the  cytoplasm 
faintly  gray  or  orange.  The  appearance  of  such  a  preparation  is  shown 
in  fig.  45.  A  similar  sharp  differential  staining  of  the  center  can  be 
achieved  by  the  use  of  iron  hsematoxylin,  the  washing  out  with  the  iron 
solution  being  prolonged  till  the  chromatin  is  colorless.  Both  nucleole 
and  center  appear  bluish  or  black  with  this  treatment.  Preparations 
made  in  this  way  show  the  persistence  of  the  center  during  the  con- 
tracted condition  of  the  chromatin  and  demonstrate  very  clearly  the 
possibility  of  differential  staining  of  the  center  by  appropriate  methods. 

It  is  clear  that  this  contracted  stage  of  the  chromatin  elements  is 
identical  with  the  synapsis  stage  in  the  spore  mother  cells  of  the  higher 
plants.  Here,  as  there,  it  is  probably  associated  with  an  interaction  and 
combination  of  the  chromosomes  prior  to  reduction,  and  the  evidence 
from  the  attachment  of  the  chromatin  threads  to  the  center  is  practi- 
cally conclusive  that  the  combination  occurs  by  the  union  of  the  chro- 
matin threads  in  pairs  side  by  side.  We  shall  find  that  the  succeeding 
spirem  stage  shows  chromatin  strands  of  unusual  thickness  and  density. 

The  contracted  condition  of  the  chromatin  is  followed  by  a  loosen- 
ing up  of  the  mass  and  a  transition  to  a  very  strongly  marked  spirem 
stage  in  the  preparation  for  the  first  division.  As  the  chromatin  mass 
spreads  out  again  into  the  antipolar  region,  and  loosens  up,  it  frequently 
appears  for  a  time  as  if  reticulated  (fig.  46).  Very  soon,  however,  the 
threads  become  more  distinct,  their  apparent  anastomoses  largely  disap- 
pear, and  they  form  an  irregular  cone,  with  its  apex  in  the  central  body 
(fig.  47),  such  as  we  found  prior  to  the  nuclear  fusion.  The  threads 
at  this  stage  are,  however,  much  more  distinct  and  sharply  outlined  than 
in  the  earlier  stages.  There  also  seem  to  be  fewer  of  the  faintly  stained 
interfilar  fibrillse.  It  should  be  noted  that  all  the  nuclei  in  the  phase  of 


SPECIAL    NUCLEAR    PHENOMENA.  41 

contraction  (figs.  43-47)  are  magnified  only  by  1,000,  while  the  figures 
of  the  earlier  stages,  Nos.  31-34,  are  magnified  by  1,500. 

The  spirem  figure  becomes  still  more  definite  with  the  further 
growth  of  the  nucleus  and  the  ascus  till,  at  a  stage  when  the  penicillate 
cells  are  well  started  in  their  development  and  the  perithecium  is  nearly 
full-sized,  the  ascus  nucleus  reaches  its  full  size  and  we  get  such  spirem 
stages  as  those  shown  in  figs.  48  and  49.  The  nuclear  structures  at 
this  stage  are  very  sharply  defined.  In  these  figures  I  have  brought  all 
the  threads  into  the  plane  of  the  median  optical  section  of  the  nucleus, 
representing  those  that  lie  above  as  darker  and  those  below  as  lighter 
and  fainter.  This  makes  the  figures  appear  much  more  crowded  and 
confused  than  they  really  are  in  the  preparations.  The  center  is  a  disk 
lying  on  the  outer  surface  of  the  nuclear  membrane,  frequently  in  a 
slight  depression,  and  from  it  very  sharply  differentiated  threads  pass 
back  and  can  be  traced  with  the  greatest  ease  into  the  antipolar  region. 
There  is  practically  no  stainable  interfilar  substance;  the  threads  lie  in 
an  unstained  nuclear  cavity,  with  nothing  to  interfere  with  the  sharpness 
of  their  outlines.  As  in  earlier  stages,  two  extremes  as  to  the  distribu- 
tion of  the  threads  in  the  nuclear  cavity  can  be  distinguished.  In  the 
one  case  some  of  the  threads  follow  rather  closely  the  inner  surface  of 
the  nuclear  membrane  and  others  pass  more  directly  to  the  antipolar 
region  through  the  midst  of  the  nuclear  cavity.  This  results  in  a  fairly 
even  distribution  of  the  chromatin  material  in  the  nucleus  (fig.  49).  In 
the  other  case  all  or  most  of  the  strands  pass  from  the  central  body 
through  the  middle  of  the  nuclear  cavity,  forming  a  spreading  bundle 
or  irregular  cone  (fig.  48),  and  then  spread  out  in  the  antipolar  region. 
Frequently  at  this  stage,  as  at  earlier  stages,  the  whole  bundle  may  be 
spirally  twisted. 

The  antipolar  ends  of  the  threads  are  frequently  seen  to  be  free. 
In  other  cases  they  are  in  contact  with  each  other,  giving  the  appear- 
ance of  being  fused  or  continuous.  Not  infrequently  they  are  in  contact 
with  the  nucleole.  The  end  region  of  a  thread  may  lie  on  the  surface 
of  the  nucleole  for  a  short  distance,  but  there  is  never  any  indication 
of  a  fusion  of  the  substance  of  the  chromatin  thread  with  that  of  the 
nucleole.  The  nucleole  is  a  sharply  defined  oval  body,  and  the  ends 
of  the  threads  appear  to  be  merely  in  contact  with  it. 

Superficially,  perhaps,  the  nucleus  at  this  stage  bears  little  resem- 
blance to  the  spirems  figured  by  Rabl  and  Flemming  for  the  salamander, 
or  by  Strasburger  for  the  endosperm  nuclei  of  Fritillaria  and  the  pollen 
mother  cells  of  the  lily,  but  there  can  be  no  question  that  this  is  a  spirem 
stage  corresponding  to  spirems  in  the  cases  mentioned  and  that  in  the 


42  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

conspicuous  attachment  of  the  chromatin  threads  to  the  central  body  we 
have  a  satisfactory  explanation  of  the  unipolar  structure  of  the  spirem 
nucleus.  Flemming's  schematic  figures  of  the  spirem  of  the  sala- 
mander are,  in  the  relation  shown  between  the  center  and  the  chromatin 
loops,  strikingly  similar  to  the  figures  shown  in  the  ascus ;  but  no  such 
conspicuous  connection  between  the  center  and  the  chromatin  strands 
as  is  seen  in  the  ascus  could  be  demonstrated  by  Flemming  in  the  cells 
of  the  salamander. 

The  number  of  strands  of  chromatin  at  this  stage  can  be  determined 
with  great  certainty.  In  practically  every  figure  which  was  studied 
careful  focusing  shows  that  there  are  just  eight  threads  passing  back 
from  the  center  into  the  antipolar  region.  Near  the  center,  of  course, 
they  may  in  some  cases  overlie  and  obscure  each  other,  but  by  tracing 
them  back  a  short  distance  toward  the  antipolar  region  the  number  can 
be  made  out  with  unfailing  regularity.  The  number  of  these  strands 
coincides,  as  we  shall  see,  with  the  number  of  chromosomes  in  the  equa- 
torial plate,  and  the  conclusion  seems  entirely  certain  that  each  of  the 
strands  corresponds  to  a  single  chromosome,  and  that  thus  each  chro- 
mosome has  a  permanent  attachment  to  the  central  body. 

A  further,  rather  long,  period  intervenes  between  the  stage  of  the 
fully  developed  spirem  and  the  equatorial  plate  of  the  first  division. 
The  perithecium  and  the  asci  continue  to  grow  slowly  in  size,  but  more 
marked  than  the  growth  in  size  at  this  period  is  the  differentiation 
which  occurs  in  the  perithecial  cells.  The  penicillate  cells  proceed  to 
their  fullest  size  and  differentiation  as  described  above.  The  append- 
ages are  developed  and  the  differentiation  of  the  outer,  middle,  and 
inner  zones  of  the  perithecial  envelopes  becomes  more  apparent. 

The  nucleus  remains  for  some  time  at  the  base  of  the  ascus,  where 
the  latter  narrows  to  form  the  short  stalk;  but  as  it  passes  on  in  its 
development  toward  the  formation  of  the  chromosomes  and  the  spindle, 
it  generally  migrates  to  a  region  higher  up  and  nearer  the  middle  of  the 
enlarged  portion  of  the  ascus.  The  further  differentiation  of  the  chro- 
mosomes now  continues.  The  process  seems  to  be  as  follows :  A  more 
densely  staining  portion  of  each  thread  becomes  differentiated  at  some 
point  in  its  length,  forming  an  elongated  and  bent  rod-shaped  body, 
which  is  to  become  the  chromosome.  The  process  of  differentiation 
seems  to  consist  in  the  drawing  together  and  the  aggregation  at  some 
point  of  the  more  densely  staining  constitutents  of  the  strand.  At  the 
same  time  a  contraction  or  shortening  of  the  whole  thread  occurs.  The 
segregation  of  the  more  stainable  portions  of  the  thread  into  the  chro- 
mosomes leaves  an  achromatic  filament  or  bundle  of  fibrillae  connecting 


SPECIAL    NUCLEAR    PHENOMENA.  43 

the  chromosome  to  the  pole.  The  process  is  analogous  to  the  short- 
ening of  the  chromatin  thread,  which  is  common  in  the  higher  plant 
and  animal  cells  in  the  prophases,  but  differs  from  it  in  the  fact  that  the 
chromosomes  are  throughout  the  process  so  conspicuously  attached  to 
the  pole.  It  is  generally  agreed  that  the  chromatin  thread  in  the  higher 
plants  consists  of  two  substances;  but  the  conspicuous  separation  of 
these  two  constituents  during  the  shortening  of  the  chromatin  thread 
seems  to  be  peculiar  to  the  mildew.  Still  it  is  to  be  remembered  also 
that  there  is  in  many  cases — for  example,  in  the  pollen  mother  cells  of 
the  larch — at  about  this  stage  a  large  increase  of  linin  fibrils  in  the 
nuclear  cavity,  and  it  is  at  least  possible  that  these  fibrils  arise  from  the 
spirem  thread  in  the  process  of  the  differentiation  of  the  chromosomes. 
The  chromosomes  appear  now  as  oblong  or  irregular  bodies  connected 
to  the  central  body  by  fine  pale-blue  stained  or  grayish  fibers.  They 
are  distributed  irregularly  in  the  nuclear  cavity,  and  in  a  polar  view  of 
the  nucleus  may  appear  as  if  supported  in  an  anastomosing  reticulum. 
They  may  be  pressed  closely  against  the  nuclear  membrane  in  some 
cases,  and  in  others  they  may  lie  in  the  central  region  of  the  nuclear 
cavity  or  may  be  closely  pressed  against  the  nucleole. 

The  spindle  is  formed  in  Phyllactinia  essentially  as  I  have  described 
for  Erysiphe  (38).  The  central  body  divides  and  the  daughter  centers 
migrate  away  from  each  other  on  the  surface  of  the  nuclear  membrane. 
In  Erysiphe  and  many  Discomycetes  the  nuclear  membrane  remains 
intact  till  the  diaster  stage.  In  Phyllactinia,  at  least  in  some  cases,  it 
may  disappear  much  earlier.  The  separation  of  the  daughter  centers 
divides  the  achromatic  filaments  which  connect  the  chromosomes  to  the 
central  body  into  two  cones  or  bundles.  This  process  seems  also  to 
bring  the  chromosomes  farther  and  farther  into  the  antipolar  region 
of  the  nucleus,  where  they  form  a  rather  dense  group  connected  by 
broad  bundles  of  fibers  with  the  daughter  centers  (fig.  52) .  The  bundles 
or  cones  of  fibers  are  distinct  until  near  their  antipolar  ends,  where  they 
cross  and  interlace  in  connecting  with  the  chromosomes. 

This  figure  resembles  that  of  Hermann  (43,  Taf.  31,  figs.  8-9),  in 
which  the  mantle  fibers  extend  from  the  spindle  poles  toward  the  chro- 
mosomes. There  is,  however,  this  essential  difference,  that  according 
to  Hermann  the  mantle  fibers  are  at  this  stage  for  the  first  time  extend- 
ing toward  and  becoming  connected  with  the  chromosomes,  while  (as 
we  have  seen  in  the  ascus)  the  chromosomes  have  been  continuously 
attached  to  the  centers  through  all  the  preceding  stages  of  nuclear  devel- 
opment. Whether  there  is  longitudinal  splitting  of  the  individual  fibers, 
or  whether  they  are  merely  separated  into  two  groups,  I  have  been 


44  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

unable  to  determine.  In  the  case  of  reduction  divisions  it  would  seem 
probable  that  the  fibers  merely  separate  in  two  groups.  There  is  no 
trace  of  a  direct  connection  between  the  centers  as  they  separate.  There 
is  no  evidence  of  the  existence  of  a  differentiated  central  spindle  at  this 
stage.  As  I  have  suggested  (38)  for  Erysiphe,  it  is  possible  that,  for 
a  time  at  least,  the  presence  of  the  nuclear  membrane  makes  a  central 
spindle  unnecessary.  As  to  the  permanence  of  the  nuclear  membrane 
and  the  absence  of  a  central  spindle  in  the  early  stages  of  the  separation 
of  the  centers,  the  conditions  in  the  ascus  are  similar  to  those  in  the 
spindle  formation  in  the  cleavage  of  the  egg  of  the  trout,  whitefish, 
and  many  invertebrate  animals  in  which  the  centers  migrate  to  opposite 
poles  of  the  nuclei  before  the  nuclear  membranes  disappear.  It  has, 
however,  been  rather  generally  assumed  by  students  of  karyokinesis  in 
these  eggs  that  the  connection  of  the  centers  to  the  chromosomes  is 
established  by  the  growth  of  spindle  fibers  into  the  nuclear  cavity  after 
the  centers  have  reached  their  position  at  the  poles.  It  is  interesting 
to  note  in  this  connection,  however,  that  Janssens  (49)  claims,  on  the 
basis  of  an  investigation  of  the  spindle  formation  in  Triton,  that  even 
here  no  central  spindle  is  formed  between  the  separating  centers  as 
described  by  Hermann  (43)  for  the  salamander. 

The  centers  continue  to  separate  until  they  have  passed  through 
an  entire  half-circle  and  come  to  lie  opposite  each  other,  forming  the 
poles  of  the  spindle.  The  fibers  attached  to  the  chromosomes  have 
shortened  at  the  same  time  and  have  drawn  the  chromosomes  up  into 
the  middle  region  between  the  poles  (figs.  53,  54).  The  nuclear  mem- 
brane may  be  still  intact  in  some  cases  (fig.  67^)  at  this  stage  in  Phyl- 
lactinia,  as  it  regularly  is  in  Erysiphe.  In  other  cases  it  seems  to  have 
entirely  disappeared  (figs.  53,  54,  670).  A  remnant  of  the  nucleole 
may  still  be  present  at  the  equatorial  plate  stage  (fig.  53). 

The  chromosomes  are  oblong  or  oval  bodies  in  the  equatorial  plate 
stage  and  stand  frequently  in  a  radial  position  on  the  spindle — that  is, 
attached  to  the  spindle  fibers  at  one  end  and  with  their  long  axes  at 
right  angles  to  the  long  axes  of  the  spindle.  They  are  small  as  com- 
pared with  the  size  of  the  spindle  and  generally  lie  entirely  free  from 
each  other,  so  that  they  can  be  very  readily  counted  (fig.  53).  The 
number  is  quite  regular  and  corresponds  with  the  number  of  strands 
attached  to  the  central  body  in  the  spirem  stage.  These  figures  agree 
with  those  I  have  already  published  for  other  Ascomycetes  in  showing 
the  incorrectness  of  Maire's  (61)  and  Dangeard's  (21)  claim  that  the 
Ascomycetes  have  regularly  four  chromosomes.  The  spindle  fibers 


SPECIAL    NUCLEAR    PHENOMENA.  45 

now  stain  more  deeply  than  in  the  prophase  stages  and  appear  blue  or 
violet  in  the  triple  stain. 

In  describing  and  figuring  the  three  successive  divisions  of  the 
nucleus  of  the  ascus  I  have  had  a  great  abundance  of  figures  at  my 
disposal  and  have  chosen  to  select  those  stages  in  each  division  which 
mutually  supplement  each  other  rather  than  to  give  a  full  series  of 
stages  in  any  one  division.  In  this  way  the  evidence  that  the  number 
of  chromosomes  is  the  same  throughout  and  that  the  central  bodies  are 
present  at  each  stage  is  brought  out  more  clearly. 

If  we  compare  the  chromosomes  in  the  equatorial  plate  with  the 
spirem  strands  in  the  prophases  we  get  evidence  of  a  great  reduction 
in  volume.  This  may,  of  course,  be  partially  due  to  condensation  of 
their  substance,  although  in  reality  the  spirem  strands  (figs.  48,  49) 
appear  quite  as  dense  as  do  the  chromosomes  in  the  equatorial  plate. 
However,  as  we  have  seen,  the  formation  of  the  chromosome  from  a 
strand  of  the  spirem  consists  in  the  segregation  of  two  substances  pres- 
ent in  the  spirem.  The  densely  staining  chromatin  aggregates  in  the 
chromosomes,  leaving  the  achromatic  portion  as  a  series  of  threads 
connecting  the  chromosomes  to  the  central  body,  and  these  threads  later 
form  the  spindle. 

Following  the  equatorial  plate  stage,  the  chromosomes  are  drawn 
back  to  the  spindle  poles,  and  during  this  process  again  their  number 
may  be  easily  determined.  Fig.  54  shows  an  early  metaphase  in  which 
the  daughter  chromosomes  are  beginning  to  separate.  In  fig.  55  it  is 
perfectly  plain  that  sixteen  daughter  chromosomes  are  being  drawn  back 
to  the  poles,  eight  on  each  half  of  the  spindle.  These  figures  are  abun- 
dant in  the  mildews  and  are  very  easily  fixed  and  stained.  Maire  has 
evidently  seen  more  than  four  chromosomes  in  many  cases  in  the 
prophases,  but  maintains  his  contention  by  calling  these  more  numerous 
bodies  "  prochromosomes  "  and  asserting  that  they  later  fuse  into  four 
true  chromosomes.  He  will  hardly  maintain,  however,  that  the  bodies 
shown  on  the  spindle  in  fig.  55  are  prochromosomes,  and  there  can  be 
no  question  that  there  are  at  least  eight  of  them  on  each  half  of  the 
spindle.  With  poor  fixation  it  is  possible  to  find  the  chromosomes  of 
the  mildew  fused  together  into  irregular  masses,  as  may  happen  also  in 
the  pollen  mother  cells  of  the  larch  or  lily,  and  it  is  doubtless  such  cases 
of  poor  fixation  which  have  misled  Maire  and  Dangeard.  The  polar 
asters  at  these  stages  are  very  strongly  developed,  and  it  is  apparent 
that  some  of  the  astral  rays  extend  to  the  plasma  membrane  of  the  ascus. 

The  central  spindle  fibers  left  after  the  chromosomes  have  reached 
the  poles  seem  to  disintegrate  and  pass  over  into  the  general  cytoplasmic 


46          SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

substance  (figs.  56,  57).  Frequently  they  may  be  much  elongated  by 
further  separation  of  the  young  daughter  nuclei,  but  this  is  not  neces- 
sary to  their  disappearance. 

The  chromosomes  are  next  found  loosely  aggregated  at  the  poles 
and  still  plainly  connected  to  the  central  body  by  the  fibers  which  drew 
them  back  from  the  equatorial  plate  region  (fig.  56).  This  results  in 
a  sort  of  diaster  stage  (fig.  57),  though  the  significance  of  the  name 
does  not  appear  in  any  conspicuous  arrangement  of  the  chromosomes. 

A  nuclear  membrane  is  next  formed,  close  in  to  the  surface  of  the 
chromosomes  at  first,  but  soon  expanding  so  that  more  or  less  clear 
space  appears  around  the  mass.  The  chromosomes  themselves  seem 
to  be  the  cause  of  this  enlargement.  They  grow  in  length  backward 
from  the  center,  at  the  same  time  swelling  and  becoming  somewhat 
irregular  and  knotted  (fig.  58).  As  a  result  we  get  at  once  a  rough 
duplicate  (fig.  59)  of  the  spirem  stage  of  the  prophases.  From  the 
central  body  coarse,  irregular  threads  are  seen  extending  back  into  the 
enlarging  nuclear  cavity. 

I  have  studied  these  stages  carefully,  and  it  seems  very  clear  that 
we  have  here,  in  reverse,  the  same  process  by  which  the  chromosomes 
were  segregated  out  of  the  spirem-strands  of  the  prophases.  The  sub- 
stance of  the  chromosome  is  being  redistributed  in  the  rapidly  growing 
achromatic  linin  substance.  The  general  resemblance  between  the  con- 
ditions in  figs.  58-59  and  47-48  is  certainly  noteworthy  and  shows  clearly 
that  the  chromosomes  in  passing  over  into  the  so-called  resting-stage 
in  the  reconstitution  of  the  daughter  nuclei  do  not  lose  their  connection 
with  the  central  body. 

As  the  reconstitution  of  the  daughter  nuclei  progresses,  the  nucle- 
oles  reappear  and  the  distribution  of  the  chromatin  becomes  progress- 
ively more  irregular.  The  strands  become  longer  and  apparently  may 
become  more  or  less  connected  by  anastomosing  fibrillae.  The  connec- 
tion with  the  central  body  is,  however,  still  perfectly  definite  and  con- 
spicuous (figs.  60,  61).  The  irregularity  of  the  strands  may  be  inter- 
preted as  due  to  a  diminished  tension  in  their  connection  with  the  central 
body  as  compared  with  the  prophase  stages.  Ultimately  the  chromatin 
may  become  quite  evenly  distributed  through  the  nuclear  cavity,  and 
from  the  polar  view  appears  much  like  an  ordinary  reticulum.  But  the 
constant  conspicuous  attachment  of  the  strands  with  the  central  body 
is  maintained  even  though,  as  is  quite  commonly  the  case,  the  nucleus 
projects  on  that  side  in  a  short  cone  or  papilla  (figs.  59-61). 

As  in  the  nuclei  of  the  ascogenous  hyphae,  the  attachment  of  this 
apparent  reticulum  to  the  center  becomes  especially  conspicuous  if  the 


SPECIAL    NUCLEAR    PHENOMENA.  47 

reticulum  is  for  any  reason  drawn  together  and  away  from  the  nuclear 
membrane.  In  such  cases  the  chromatin  threads  are  always  attached 
to  the  center,  though  they  may  be  drawn  away  from  the  membrane 
everywhere  else.  This  condition  is  also  conspicuous  in  the  figures  of 
Erysiphe  which  I  have  already  published.  The  polar  asters  through 
these  stages  are  very  sharply  differentiated,  the  fibers  extending  in 
some  cases  almost,  if  not  entirely,  to  the  plasma  membrane  of  the  ascus 
(figs.  55-61). 

The  daughter  nuclei  formed  by  the  division  of  the  primary  nucleus 
of  the  ascus,  as  just  described,  never  grow  to  the  size  of  the  mother 
nucleus.  Their  volume  is  apparently  not  more  than  one-half  that  of 
the  primary  nucleus.  As  a  rule  they  divide  again  immediately,  though 
in  some  cases  apparently  a  considerable  period  may  intervene. 

Fig.  62  shows  an  early  stage  of  the  spirem  of  the  nucleus  in  the 
binucleated  stage  of  the  ascus ;  and  fig.  63  a,  b  shows  a  stage  in  the  sepa- 
ration of  the  daughter  centers  and  the  formation  of  the  spindle.  The 
chromosomes  appear  as  oblong,  densely  stained,  bent  or  irregular  bodies 
in  a  fairly  dense  group  in  the  antipolar  region.  The  group  was  cut  in 
two  in  sectioning  and  is  reproduced  in  the  two  figures  (63^  b}.  The 
halves  of  the  spindle  appear  as  broad  series  of  fine  achromatic  fibers 
and  the  centers  have  their  characteristic  flat,  disk-shaped  form.  The 
stage  is  a  little  earlier  than  that  shown  in  fig.  52  for  the  first  division. 
It  differs  in  that  the  nuclear  membrane  is  in  this  case  still  partially 
present,  though  breaking  down  in  certain  portions.  Fig.  64  shows  a 
somewhat  later  stage,  in  which  the  centers  are  farther  apart  and  the 
halves  of  the  spindle  diverge  at  a  correspondingly  greater  angle.  In 
this  case  the  polar  asters  appear  as  well-developed  systems  of  fibers 
radiating  from  the  central  bodies  into  the  cytoplasm. 

Fig.  65  shows  the  two  nuclei  of  the  ascus  in  the  equatorial  plate 
stage.  The  upper  spindle  lies  in  the  plane  of  the  section  and  the  lower 
is  cut  through  obliquely,  so  that  only  one  pole  and  one  half  of  the  spindle 
appear  in  the  section.  As  is  to  be  seen,  eight  chromosomes  are  present 
at  this  stage  also.  In  Phyllactinia,  as  in  all  the  Ascomycetes  I  have 
studied,  the  number  of  chromosomes  remains  the  same  through  all  three 
divisions  of  the  primary  nucleus  of  the  ascus.  Fig.  66  shows  a  further 
stage  in  the  division  of  the  two  nuclei.  One  spindle  lies  in  the  long 
axis  of  the  ascus  and  the  other  almost  transversely  and  in  the  upper  end 
of  the  ascus.  As  a  rule  only  two  spores  are  formed  in  the  ascus  of 
Phyllactinia;  and  a  study  of  the  further  stages  shows  that  both  the 
daughter  nuclei  produced  from  this  transverse  spindle  will  remain  in 
the  upper  end  of  the  ascus  and  fail  to  become  centers  for  spore  forma- 


48  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

tion.  In  this  upper  spindle  the  chromosomes  are  a  little  nearer  the 
poles  than  in  the  lower  one,  but  in  both  either  seven  or  eight  chromo- 
somes can  be  made  out  in  each  group. 

Figs.  67  a,  b  show  three  nuclei  from  an  ascus  in  the  four-nucleated 
stage.  They  are  all  in  the  stage  of  division  when  the  daughter  chro- 
mosomes are  just  separating  out  of  the  equatorial  plate.  Fig.  67  a 
shows  one  profile  and  one  polar  view  of  the  spindles  which  lie  at  the 
upper  end  of  the  ascus  and  are  destined  to  form  supernumerary  nuclei. 
Fig.  67  b  shows  a  somewhat  larger  spindle  figure  which  lies  near  the 
center  of  the  ascus.  It  is  probable  that  both  nuclei  formed  from  it  will 
become  centers  for  spore  formation.  In  this  division  also  the  chromo- 
somes can  be  counted  with  perfect  certainty  as  they  are  drawn  back  to 
the  poles.  Fig.  68  shows  two  nuclei  at  this  stage,  and  in  each  of  the 
four  daughter  groups  seven  or  eight  chromosomes  can  be  counted. 

In  both  the  second  and  third  divisions  it  is  plain  that  the  central 
body  continues  in  the  same  relation  to  the  chromatin  as  in  the  first 
division  and  the  fusions  which  preceded  it.  The  figures  in  the  two  and 
four  nucleated  stages  are  not  so  favorable  for  counting  the  number  of 
strands  in  the  spirem  stage  of  the  prophases  by  reason  of  their  greatly 
diminished  size,  but  as  to  the  main  fact,  that  the  central  body  maintains 
a  continuous  connection  with  the  chromatin,  just  as  in  the  first  division, 
the  evidence  is  perfectly  conclusive. 

As  noted  above,  only  two  spores  are  formed  as  a  rule  in  the  asci 
of  Phyllactinia.  This  leaves  six  supernumerary  nuclei  which  disinte- 
grate in  the  epiplasm.  Generally  these  supernumerary  nuclei  at  the 
time  of  spore  formation  are  all  in  the  peripheral  end  of  the  ascus,  while 
the  two  nuclei  which  are  to  become  the  centers  of  the  two  spores  are 
rather  above  the  middle  of  the  ascus.  The  supernumerary  nuclei  fre- 
quently are  pressed  against  the  wall  of  the  ascus,  with  their  central 
bodies  on  the  side  next  the  wall.  Later  several  of  these  supernumerary 
nuclei  are  frequently  found  lying  in  a  bunch  free  in  the  cytoplasm. 

The  process  of  spore  formation  is  especially  well  shown  in  the  asci 
of  Erysiphe  cichoracearum,  and  I  have  included  for  these  stages  some 
figures  from  this  species  with  those  from  Phyllactinia.  Figs.  80  and  81 
are  from  £.  communis.  The  polar  aster  from  the  third  division  persists 
in  all  the  eight  nuclei  for  some  time,  but  is  most  conspicuous  in  the  case 
of  the  nuclei  which  are  to  be  inclosed  in  spores  (fig.  69).  A  beak  is 
next  pushed  or  pulled  out  from  the  nucleus,  which  is  generally  already 
pear-shaped,  as  in  the  corresponding  stages  in  the  earlier  divisions 
(figs.  70,  7I>72)- 


SPECIAL    NUCLEAR    PHENOMENA.  49 

The  metamorphosis  of  the  polar  aster  in  Phyllactinia  and  Brysiphe 
cichoracearum  is  entirely  similar  to  that  in  Brysiphe  communis.  In 
view  of  the  above-described  facts  as  to  the  connection  between  chro- 
matin  and  central  body  at  other  stages,  it  is  interesting  to  note  that  in 
the  protrusion  of  the  nuclear  beak  the  same  condition  comes  most  strik- 
ingly to  view.  The  beak  is  not  merely  an  extension  of  the  nuclear 
membrane,  but  the  chromatin  is  also  pulled  out  as  slender  strands  in  the 
beak  and  maintains  its  connection  with  the  central  body  through  the 
whole  process  of  the  delimitation  of  the  spore.  This  is  very  clearly 
shown  in  both  Erysiphe  and  Phyllactinia  (figs.  70-78). 

The  central  body  on  the  apex  of  the  beak  is  very  broad  and  flat  in 
Phyllactinia  (fig.  72) .  Its  diameter  is  apparently  greater  than  that  of  the 
outer  portion  of  the  beak.  I  have  occasionally  found  cases  in  which  the 
central  body  had  divided  at  the  end  of  the  beak  and  the  fibers  were  also 
separated  into  two  systems  (fig.  80).  Whether  this  condition  would  be 
followed  by  normal  spore  formation  I  have  not  determined.  It  shows  the 
capacity  of  the  centers  to  divide  and  seems  to  indicate  that  such  division 
separates  the  fibers  into  two  groups  without  splitting  each  fiber. 

The  process  by  which  the  beak  is  formed  on  the  nucleus  is  not  easy 
to  understand.  I  have  elsewhere  discussed  (38)  the  possible  methods 
by  which  such  a  beak-like  elongation  may  be  pulled  or  pushed  out  from 
the  surface  of  the  nucleus.  My  observations  on  Phyllactinia  and 
B-  cichoracearum  incline  me  more  strongly  to  the  view  that  it  is  formed 
by  the  activity  of  the  aster  rather  than  by  any  spontaneous  change  of 
form  in  the  relatively  inert  nuclear  mass. 

In  B.  cichoracearum  it  seems  plain,  as  I  found  was  the  case  some- 
times in  Lachnea  and  Pyronema,  that  in  some  cases,  at  the  time  of  the 
formation  of  the  beak,  the  center  and  aster  may  be  in  close  contact  with 
the  plasma  membrane  of  the  ascus  (figs.  69,  73,  74),  just  as  is  quite 
regularly  the  case  with  the  supernumerary  nuclei  (figs.  70,  73,  81). 
The  folding  over  of  the  rays  may  begin  while  the  centers  are  in  this 
position  and  may  seem  to  be  a  result  of  the  flattening  of  the  aster 
against  the  membrane  of  the  ascus  (figs.  73,  74). 

In  Phyllactinia  the  beaked  nucleus  and  aster  seem  much  more  com- 
monly to  lie  free  in  the  cytoplasm  from  the  start  (figs.  70,  71,  72). 
Occasionally,  even  when  the  center  is  quite  distant  from  the  plasma 
membrane,  a  broad  depression  is  found  in  the  latter  just  opposite  the 
center,  as  if  the  astral  rays  were  attached  to  it  and  by  contraction  had 
pulled  it  away  from  the  cell-wall  (fig.  71).  In  the  later  stages  in  all 
cases  the  whole  system  is  found  lying  free  in  the  cytoplasm  and  gener- 
ally at  some  distance  from  the  wall  of  the  ascus.  It  is  quite  possible  that 


c;O  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

t* 

while  in  contact  with  the  plasma  membrane  of  the  ascus  some  of  the 
material  of  the  latter  may  pass  over  into  the  rays  and  thus  aid  in  forming 
the  membrane  of  the  spore.  There  is,  however,  no  break  in  the  plasma 
membrane  of  the  ascus  as  a  result  of  any  such  possible  participation 
in  the  formation  of  the  new  spore  membrane.  The  membrane  of  the 
ascus  remains  as  a  continuous  envelope  of  its  cytoplasm,  and  there  is 
no  apparent  loss  of  turgidity  in  the  latter.  In  Phyllactinia  also  it  is 
quite  common  to  find  the  cytoplasm  very  loose  and  showing  large  vacu- 
oles  in  the  neighborhood  of  the  beaked  nuclei  (figs.  70,  72).  These 
spaces  may  represent  the  remains  of  the  nuclear  cavity  of  the  preceding 
mother  nucleus.  This  looser  cytoplasm  is  sharply  bounded  by  the  inner 
rays  of  the  aster  (figs.  70,  72). 

The  stages  in  the  process  of  the  folding  over  of  the  rays  and  their 
union  to  form  the  plasma  membrane  of  the  spore  are  very  well  shown 
in  E.  cichoracearum.  The  rays  become  elongated  during  the  process 
by  growth  which  apparently  proceeds  from  the  central  body  outward, 
and  at  the  same  time  they  fold  over  and  combine  side  by  side  to  form  a 
continuous  broad,  umbrella-shaped  membrane  (figs.  74,  81).  Sometimes 
the  rays  on  one  side  seem  to  be  in  advance  of  those  on  the  other  in  the 
process  of  inclosing  the  spore-mass  (fig.  73).  If,  in  folding  over  and 
elongating,  the  rays  of  one  center  come  in  contact  with  those  of  another, 
they  tend  to  fuse,  at  least  temporarily  (fig.  74).  Later,  however,  they 
must  separate  again,  since  one  almost  never  finds  spores  with  two  nuclei, 
while  such  conditions  as  those  shown  in  fig.  74  are  not  uncommon. 

That  the  rays  actually  combine  to  form  a  membrane  in  these  early 
stages  is  shown  in  Phyllactinia,  as  in  E.  communis,  by  the  fact  that  the 
polar  region  of  the  spore  may  draw  away  from  the  adjacent  cytoplasm 
as  a  result  of  fixation  before  the  spore  is  entirely  delimited.  The  broad, 
umbrella-shaped  membrane  shown  in  figs.  74  and  81  gradually  closes  in 
to  form,  by  further  marginal  growth,  the  ellipsoidal  plasma  membrane 
of  the  spore  (fig.  75).  The  whole  spore  body  is  cut  out  of  the  previ- 
ously undifferentiated  cytoplasm  of  the  ascus  by  the  formation  of  a  new 
plasma  membrane  derived  from  the  fibers  of  the  polar  aster  and  without 
the  deposition  of  a  cellulose  wall.  In  this  case,  as  in  animals  and  the 
higher  plants,  the  process  of  cell  division  consists  in  the  formation  of 
new  plasma  membranes  which,  in  the  latter  at  least,  originate  as  a  so- 
called  cell  plate  from  the  fibers  of  a  portion  of  the  karyokinetic  figure. 

Such  a  process  of  migration  of  the  astral  fibers  demands  the 
assumption  that  they  are  contractile  elements  comparable  to  cilia,  even 
though  we  have  not  as  yet  sufficient  data  on  which  to  carry  out  such  a 
comparison  in  every  detail.  The  abundant  evidence  which  has  accu- 


SPECIAL    NUCLEAR    PHENOMENA.  51 

mulated  in  recent  years,  in  both  animals  and  plants,  that  the  ciliary 
apparatus  of  the  male  cell  arises  from  a  more  or  less  modified  central 
body,  is  strong  ground  for  the  acceptance  of  this  view.  Further,  the 
process  of  transformation  of  the  polar  aster  into  an  ellipsoidal  mem- 
brane is  to  be  compared  with  other  cases  of  intra-cytoplasmic  migra- 
tions, such  as  those  of  the  central  spindle  fibers  in  building  the  cell 
plate  and  the  migrations  of  the  asters  of  the  sperm  cell  in  the  animal 
egg  in  the  processes  of  fertilization.  Though  the  resemblances  in  all 
these  processes  are  not  yet  clear,  we  certainly  have  sufficient  ground  for 
assuming  that  the  fibrous  material  concerned  in  them  all  constitutes  a 
specially  differentiated  material  of  the  cell — the  kinoplasm. 

That  the  spore  as  at  first  formed  has  no  cellulose  wall  is  well  shown 
in  cases  in  which  it  is  plasmolyzed  in  fixation.  In  such  cases  the  sepa- 
ration of  the  spore-plasm  from  the  epiplasm  is  complete  and  there  is 
no  trace  of  a  cell-wall  in  the  cleft  so  formed.  The  surface  of  the  plas- 
molyzed spore  shows  the  same  continuity  as  we  find  in  the  surface  of 
the  protoplast  of  the  ascus,  and  there  can  be  no  doubt  that  the  spore 
boundary  formed  by  the  fibers  of  the  polar  aster,  even  at  this  early  stage, 
is  essentially  similar  in  its  nature  to  the  layer  bounding  the  entire  ascus 
or  other  cells  of  the  mycelium. 

Whether  the  material  of  the  fibers  has  undergone  a  chemical  change 
in  forming  this  continuous  membranous  layer  is  difficult  to  determine. 
That  their  staining  reactions  change  somewhat  seems  fairly  evident  in 
most  cases.  As  fibers  of  the  polar  aster  they  take  the  blue  color  in  the 
triple  stain.  On  the  surface  of  the  completely  delimited  spore  it  is 
difficult,  if  not  impossible,  to  distinguish  a  layer  differently  stained 
from  the  spore-plasm  which  it  surrounds.  Still  occasionally,  in  the 
triple  stain,  this  layer  does  show  a  bluish-gray  tint  somewhat  clearly 
differentiated  from  the  gray  or  faint  orange  of  the  inner  spore-plasm. 
If,  as  Overton  (73,  74)  concludes,  the  outer  layer  of  the  protoplast  is 
a  cholesterin-like  substance  or  is  impregnated  with  such  a  substance, 
it  must  probably  be  assumed  that  the  kinoplasmic  fibers  undergo  a 
decomposition  in  forming  the  limiting  layer  of  the  spore.  On  the  other 
hand,  the  fact  that  these  fibers  maintain  their  identity  and  do  not  mingle 
with  or  dissolve  in  the  cytoplasm  about  them  while  they  form  the  polar 
aster  is  sufficient  evidence  that  their  substance  is  capable,  without  chem- 
ical change,  of  forming  an  osmotic  layer  sufficiently  differentiated  to 
separate  the  spore-plasm  from  the  remaining  cytoplasm,  at  least  to  an 
extent  which  would  permit  the  plasmolyzing  of  the  spore  mass. 

In  the  case  of  these  preparations  with  shrunken  spores  we  can  make 
out,  by  careful  observation,  in  some  cases,  an  interesting  difference 


C2  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

*J 

between  the  surface  of  the  spore  and  that  of  the  epipiasm  from  which 
it  is  drawn  back.  The  surface  of  the  spore  is  plainly  smooth  and  con- 
tinuous, while  that  of  the  epipiasm  is  slightly  ragged  and  irregular, 
indicating  that  the  spongy  cytoplasmic  reticulum  has  been  cut  through 
without  closing  the  interstices  of  the  epipiasm,  either  by  the  rounding 
out  of  reentrant  cavities  by  surface  tension  or  by  the  deposit  of  a  surface 
layer  of  material  to  close  such  openings.  The  spore-plasm  is  evidently 
inclosed  and  smoothly  enveloped  by  the  material  of  the  polar  fibers, 
while  no  such  substance  has  been  deposited  on  the  corresponding  sur- 
face of  the  epipiasm. 

After  the  spore  is  completely  inclosed  the  remnant  of  the  fibers 
disappears  from  the  region  about  the  central  body  (fig.  76).  The  latter 
then  breaks  away  from  the  plasma  membrane  and  the  nucleus  gradually 
regains  its  spherical  or  oval  shape  by  drawing  in  the  beak-like  prolon- 
gation. The  stages  in  this  process  are  very  well  shown  in  Phyllactinia 
(figs.  77,  78).  The  central  body  comes  thus  to  occupy  its  old  position 
on  the  surface  of  the  spherical  nucleus.  The  chromatin  is  apparently 
at  this  stage  an  irregular  reticulum,  but  is  always  attached  to  the  central 
body.  It  is  frequently  drawn  back  from  the  nuclear  membrane  into  an 
irregular  mass  on  all  sides,  except  where  it  is  attached  to  the  central 
body  (fig.  79).  Later  a  wall  is  built  around  the  spore  and  a  resting 
condition  ensues  which  lasts  till  the  bursting  of  the  perithecia  in  the 
following  spring.  The  connection  of  chromatin  and  center  can  be 
observed  in  favorable  preparations  in  the  fully  matured  spores,  but  these 
stages,  owing  to  the  presence  of  the  spore  wall,  are  less  easily  fixed  than 
the  earlier  ones.  In  the  fully  ripened  condition  a  considerable  amount 
of  reserve  material  is  deposited  in  the  spore-plasm,  which  thus  becomes 
quite  different  in  its  composition  from  the  surrounding  epipiasm. 

The  germination  of  the  ascospores  into  a  vegetative  mycelium  com- 
pletes the  life  cycle  of  the  mildew.  The  nuclei  of  the  mycelial  hyphae, 
as  already  described,  show  the  same  polar  structure,  with  the  chromatin 
directly  connected  with  the  central  body,  which  we  have  traced  through 
the  stages  in  the  development  of  the  ascocarp.  We  thus  have  a  fairly 
continuous  account  of  the  existence  of  the  central  body  and  the  main- 
tenance of  its  connection  with  the  material  of  the  chromosomes  through 
two  nuclear  fusions  in  the  oogonium  and  in  the  young  ascus,  through 
a  series  of  divisions  in  the  ascogenous  hyphae,  and  the  triple  division  in 
the  ascus,  and  finally  through  the  formation  of  the  ascospores  by  free 
cell  formation.  This  constitutes  the  longest  and  most  varied  series  of 
stages  through  which  the  central  body  of  a  plant  has  yet  been  traced. 


CENTRAL    BODY    IN    PHYLLACTINIA.  53 


THEORETICAL   DISCUSSION. 

THE    CENTRAL    BODY    IN    PHYLLACTINIA. 

We  find  in  the  above-described  series  of  nuclear  fusions  and 
divisions  not  alone  the  persistency  of  a  central  body  as  such,  but  a  center 
that  remains  throughout  in  intimate  and  organized  connection  with  the 
chromatin  content  of  the  nucleus.  The  center  is  not  a  naked  granule 
or  centrosphere  which  can  be  distinguished  only  with  difficulty  from 
other  stainable  granules  in  the  neighborhood  of  the  nucleus.  It  con- 
stitutes throughout  a  point  of  attachment  for  the  elements  of  the  nucleus, 
and  in  all  the  various  modifications  which  it  and  they  undergo  in  the 
processes  of  division  and  fusion  this  relation  is  maintained  in  the  most 
definite  fashion.  The  central  body  by  its  position  determines  in  an 
important  sense  a  definite  polar  organization  on  the  part  of  the  chro- 
matin, and  thus  of  the  nucleus  as  a  whole.  At  no  stage  in  its  develop- 
ment is  the  nucleus  of  Phyllactinia  an  isotropic  or  radially  organized 
body.  In  every  stage  the  chromatin  is  definitely  attached  to  either  one 
or  two  central  bodies  on  the  periphery  of  the  nucleus.  The  nucleus  is 
hence  strictly  unipolar  throughout  its  so-called  resting  stages,  becoming 
bipolar  by  division  of  the  center  for  the  formation  of  the  two  daughter 
nuclei.  We  can  thus  distinguish  throughout  its  history  both  polar  and 
antipolar  regions  in  the  nucleus.  The  position  of  the  central  body  on 
the  nuclear  membrane  is  characteristic  of  the  fungi,  and  the  greater 
readiness  with  which  a  permanent  connection  between  the  nuclear  ele- 
ments and  the  center  can  be  demonstrated  in  them  is  no  doubt  associated 
with  this  fact. 

Further,  it  is  plain  that  the  chromatin  elements  are  throughout  defi- 
nite in  number  and  each  one  is  attached  independently  to  the  center. 
In  the  so-called  spirem  stage  each  chromatin  element  consists  of  a 
relatively  thick  thread  or  strand,  which  is  attached  at  one  of  its  ends  to 
the  center,  from  which  it  extends  back  to  the  antipolar  region  of  the 
nucleus,  where  it  ends  freely,  or  may  be  loosely  joined  to  the  other 
strands,  or  lie  in  contact  with  the  nucleole. 

In  the  reconstitution  of  the  daughter  nuclei,  from  the  diaster  stage 
on,  the  chromosomes  elongate  and  pass  back  into  their  more  diffuse 
condition  without  losing  their  connection  with  the  center;  and  even 
when  the  daughter  nucleus  passes  into  the  complete  resting  condition 
and  the  chromatin  strands  apparently  anastomose  by  fibrillar  outgrowths 


54  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

to  form  what  may  appear  to  be  a  chromatic  reticulum,  its  attachment  to 
the  central  body  is  still  conspicuous  and  indicates  that  each  strand  main- 
tains its  separate  and  individual  connection  with  the  nuclear  pole.  In 
favorable  preparations  close  analysis  of  the  resting  stages  show  consid- 
erable evidence  that  the  eight  chromosomes  are  still  to  be  differentiated 
as  constituting  the  main  strands  of  the  reticulum,  though  they  may  be 
quite  irregular  in  their  outline  and  connected  by  anastomosing  fibrillse. 
The  central  bodies  are  thus  seen  to  be  permanent  structures  of  the  cell 
during  both  the  dividing  and  resting  stages  of  nuclear  development. 

In  the  processes  of  nuclear  fusion  in  Phyllactinia  the  permanence 
of  the  central  body  is  also  evident.  In  the  vegetative  nuclear  fusions 
in  the  young  ascus  each  nucleus  has  a  conspicuous  polar  structure  and 
central  body  at  the  time  when  the  nuclear  membranes  break  down  and 
the  nuclear  cavities  combine.  The  fusion  nucleus  thus  formed  has  for  a 
time  two  centers  and  two  independent  systems  of  strands  of  chromatin. 
These,  however,  gradually  approach  and  combine  into  a  single  centered 
system  with  one  central  body.  The  centers  fuse  and  the  chromatin 
strands  combine,  so  that  the  fusion  nucleus  has  one  central  body  and 
the  same  apparent  number  of  strands  of  chromatin  as  each  of  the  nuclei 
which  combine  to  form  it.  In  the  sexual  fusion  at  the  initiation  of  the 
ascocarp  we  find  both  antheridial  and  egg  nuclei  provided  with  conspic- 
uous centers.  When  the  pronuclei  are  lying  side  by  side  in  the  egg  their 
centers  are  also  still  present.  The  small  size  of  the  nuclei  and  of  the 
whole  sexual  apparatus  at  this  stage  makes  it  difficult  to  trace  the  stages 
in  the  combination  of  the  pronuclei,  but  the  fertilized  egg-nucleus  shows 
conspicuously  a  single  center,  and  it  seems  probable  that  here,  as  in  the 
fusion  of  the  ascus,  this  center  takes  its  origin  in  the  union  of  the  centers 
of  the  fusing  nuclei.  The  permanence  of  the  centers  throughout  the 
remaining  stages  in  the  life  history  of  the  fungus,  and  especially  their 
definite  connection  with  the  chromatin  content  of  the  nucleus,  makes 
it  highly  improbable  that  either  one  of  them  should  disappear  and  be 
replaced  by  the  other  during  the  process  of  fertilization. 

In  the  process  of  spore  formation  by  free  cell  division  the  center  is 
also  constantly  and  conspicuously  present,  and  we  are  justified  in  con- 
cluding that  in  Phyllactinia  the  central  body  is  a  permanent  cell  struc- 
ture maintaining  its  identity  through  the  whole  life  history  of  the  plant, 
involving  the  varied  processes  of  nuclear  division,  nuclear  fusion,  and 
free  cell  formation.  This,  of  course,  does  not  necesarily  imply  the 
individuality  of  the  center  in  the  sense  that  it  is  to  be  considered  an 
elementary  organism  or  even  an  organ  with  complex  internal  structure, 
such  as  we  seem  bound  to  conceive  is  present  in  the  chromosome.  The 


CENTRAL  BODY  IN  PHYLLACTINIA.          55 

facts  seem  adequately  accounted  for  by  the  conception  that  the  central 
body  is  a  more  permanent  cell  element  composed  of  the  same  kino- 
plasmic  material  which  is  found  in  the  polar  rays  and  spindle  fibers. 

I  am  of  the  opinion  that  the  various  activities  shown  in  spindle 
formation  and  the  movement  of  the  chromosomes,  as  well  as  in  free  cell 
formation,  are  to  be  regarded  as  functions  of  the  individual  contractile 
kinoplasmic  fibrillae  of  the  spindle  and  asters  rather  than  of  the  centers 
in  which  these  fibers  meet  and  are  combined.  The  determination  of  a 
constant  fibrous  connection  between  the  central  body  and  the  chromo- 
somes is  a  strong  point  against  the  so-called  dynamic  theories  of  the 
centrosome.  In  such  a  system  we  have  no  need  for  the  assumption  of 
any  radially  working  force  which  goes  out  from  the  sphere  as  a  dynamic 
center.  The  motions  of  all  the  bodies  connected  with  the  center  are 
much  more  adequately  provided  for  by  the  assumption  of  contractility 
in  the  kinoplasmic  fibers  which  connect  them  to  the  center.  This  con- 
tractility is  to  be  compared  to  that  of  the  cilia  and  the  elements  of  the 
muscle  cell.  The  comparison  of  the  kinoplasmic  fibers  to  cilia  or  muscle 
elements  suggests  further  that  the  fibers  need  not  necessarily  be  arranged 
in  centered  systems,  and  indeed  we  have  abundant  evidence  in  the  higher 
plants  that  the  kinoplasmic  fibers  may  perform  their  characteristic  func- 
tions in  nuclear  and  cell  division  without  the  presence  of  a  central  body. 
Under  this  conception  of  the  central  body,  the  types  of  spindle  forma- 
tion in  the  vascular  plants  on  the  one  hand  and  in  the  fungi  and  algae 
and  the  animals  on  the  other  can  be  brought  together. 

The  conception  of  a  unipolar  structure  of  the  resting  nucleus 
plainly  can  not  apply  to  the  nuclei  of  the  higher  plants,  whose  spindles 
are  formed  from  a  perinuclear  weft  of  fibers.  Still,  it  is  by  no  means 
impossible  that  a  permanent  connection  between  the  chromatin  elements 
and  the  surrounding  material  of  the  cytoplasm  by  kinoplasmic  fibrillee 
exists  also  in  these  cases.  There  is  considerable  evidence  in  the  pro- 
phases  of  division  in  the  pollen  mother  cells  of  the  larch,  as  shown  by 
Allen  (i),  that  the  fibers  of  the  cytoplasm  which  later  form  the  spindle 
are  connected  through  the  nuclear  membrane  with  the  chromosomes. 
The  difficulty  in  understanding  how  the  daughter  chromosomes  in  turn 
become  attached  to  fibers  from  one  pole  only  of  the  spindle,  unless  the 
chromosomes  occupy  definite  regions  in  the  nucleus  and  are  perma- 
nently attached  to  the  kinoplasmic  fibers,  is  just  as  great  here  as  in  the 
case  of  the  formation  of  the  spindle  in  animal  cells.  Still,  Strasburger 
(89)  holds  that  in  the  vegetative  divisions  in  various  root  tips  the 
nuclear  membrane  disappears  at  the  poles  and  the  spindle  fibers  grow 


56  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

in  and  attach  themselves  to  the  chromosomes  or  meet  and  form  the 
central  spindle  fibers  extending  from  pole  to  pole. 

The  figures  of  Swingle  (920)  seem  decidedly  to  favor  the  view 
that  in  Stypocaulon  the  spindle  fibers  form  new  connections  with  the 
chromosomes  by  growing  in  from  the  poles  at  each  mitosis.  The  same 
is  true  of  the  spindle  formation  in  the  first  division  of  the  tetraspore 
mother  cell  of  Dictyota,  as  described  by  Mottier  (67) .  On  the  other  hand, 
the  same  author's  figures  of  spindle  formation  in  the  second  division 
seem  to  agree  closely  with  the  corresponding  stages  in  the  ascus. 

PERMANENCE  OF  THE  CHROMOSOMES. 

It  is  evident  that  the  resting  reticulum  of  the  nucleus  of  the  mildew 
contains  at  least  two  elements,  and  that  the  chromosomes  of  the  equa- 
torial-plate stage  are  formed  from  the  chromatin  of  the  resting  and 
spirem  stages  by  the  segregation  of  a  less  stainable  material — the  linin — 
and  a  denser,  more  stainable  material.  The  latter  becomes  condensed 
into  the  oblong  chromosomes  connected  to  the  central  body  by  bundles 
of  achromatic  threads.  The  formation  of  the  spirem  is  a  process  in 
which  the  principal  strands  of  the  reticulum  come  more  prominently 
into  view  as  a  result  of  their  contraction  while  attached  at  one  end  to  the 
central  body.  In  this  process  they  lose  the  fibrillae  by  which  they  were 
connected  to  form  the  resting  reticulum  and  become  sharply  defined, 
highly  stainable  threads  lying  in  an  entirely  achromatic  nuclear  sap. 

The  material  of  each  chromosome  is  still  distributed  at  the  spirem 
stage  through  the  whole  length  of  the  strand  out  of  which  it  is  later 
segregated.  The  withdrawal  of  this  chromatic  material  into  the  com- 
pact body  of  the  chromosome  leaves  the  spirem  strand  as  a  thin  achro- 
matic fiber  or  bundle  of  fibers  connecting  the  chromosomes  to  the  pole. 
Whether  in  the  division  of  the  central  body  and  separation  of  the 
daughter  centers  to  form  the  poles  of  the  spindle  the  achromatic  fibers 
are  split  longitudinally  or  are  merely  separated  into  two  bundles  is  not 
clear  from  my  preparations.  It  is,  however,  plain  that  each  chromo- 
some is  from  the  start  connected  with  both  poles,  and  that  provision  is 
thus  made  for  the  separation  of  the  daughter  chromosomes  and  their 
withdrawal  to  the  poles  of  the  spindle  to  form  the  daughter  nuclei. 

During  the  separation  of  the  daughter  centers  no  so-called  central 
spindle  is  present,  but  in  the  anaphases  central  spindle  fibers  running 
through  from  pole  to  pole  are  conspicuous;  and  they  persist  until  the 
daughter  nuclei  have  begun  their  independent  development,  being  then 
apparently  gradually  disintegrated.  How  these  central  spindle  fibers 
arise  as  distinct  from  the  fibers  which  draw  the  chromosomes  to  the 
poles  is  not  clear. 


PERMANENCE   OF   THE    CHROMOSOMES.  57 

In  the  fusion  of  the  nuclei  of  the  young  ascus  the  generally  parallel 
position  of  the  strands  of  the  two  chromatin  systems  at  the  time  the 
centers  unite  leads  naturally  to  the  assumption  that  the  individual  strands 
are  combined  side  by  side,  so  that  the  number  of  the  chromosomes  is 
not,  apparently,  doubled  in  the  fusion  nucleus.  The  fusion  of  the  male 
and  female  pronuclei  probably  proceeds  in  the  same  fashion,  so  that 
here  again  the  chromosome  number  in  the  fertilized  egg  will  not  appear 
to  be  doubled,  though  the  individual  chromosomes  must  be  regarded  as 
bivalent  structures.  The  details  of  this  fusion  I  have  not  as  yet  been 
able  to  make  out,  but,  since  the  chromatin  of  the  pronuclei  is  plainly 
attached  to  the  centers,  just  as  in  the  fusion  in  the  ascus,  it  is  probable 
that  the  method  of  combination  is  the  same  in  both. 

The  evidence  given  above,  that  the  chromosomes  are  in  continuous 
connection  with  the  central  body  in  the  resting-stages,  as  well  as  when 
dividing  and  fusing,  is  fairly  conclusive  that  in  Phyllactinia,  and  pre- 
sumably in  other  mildews,  the  chromosomes  are  permanent  cell  struc- 
tures. The  facts  which  favor  the  doctrine  that  the  chromosomes  are 
everywhere  permanent  cell  organs  have  accumulated  very  rapidly  and 
from  many  sources  in  recent  years.  This  evidence,  both  from  older 
and  more  recent  authors,  as  to  permanency  of  size,  number,  form, 
position  in  the  nucleus  of  the  whole  series  of  chromosomes,  and  the 
further  remarkable  facts  of  chromosome  differentiation  in  size,  form, 
etc.,  as  described  by  Henking  (42),  Montgomery  (66),  and  others  for 
the  accessory  chromosome,  and  by  Sutton  (92)  for  the  whole  series  of 
chromosomes  in  Brachystola,  has  all  been  fully  summarized  and  its 
significance  critically  estimated  by  Boveri  (13).  Still  more  recently 
Rosenberg  (8ia)  has  described  the  existence  of  chromosomes  of  unequal 
size  in  Listera.  The  vegetative  cells  here  show  regularly  10  large  and 
22  smaller  chromosomes. 

We  have  in  these  newer  facts  not  only  proof  of  specific  differences 
between  chromosomes,  but  indisputable  evidence  that  individual  chro- 
mosomes are  perpetuated  as  such  from  one  cell  generation  to  another. 
It  is  a  question,  however,  whether  Boveri  is  justified  in  combining  with 
the  conception  of  the  permanency  of  the  chromosomes  as  cell  structures 
the  further  doctrine  that  they  are  individual  and  elementary  organisms 
leading  a  relatively  independent  existence  in  the  cell,  and  thus  in  a 
sense  comparable  in  their  individuality  to  the  cell  itself.  It  is  doubtful 
whether  the  facts  of  permanence  in  number,  form,  position  in  the 
nucleus,  etc.,  even  suggest  any  such  conclusion. 

The  conception  of  the  cell  as  made  up  in  whole  or  part  of  more 
elementary  independent  organisms  is  not  a  necessary  conclusion  from, 


58  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

nor  to  be  confused  with,  the  conception  that  the  cell  mechanism  contains 
definite  and  permanent  parts  with  specific  functions.  It  may  even  be 
a  question  whether  it  is  advisable  to  call  such  parts  of  the  cell  organs, 
since  they  are  not  to  be  compared  morphologically  to  the  organs  of 
multicellular  plants  or  animals. 

The  most  specific  evidence  which  Boveri  advances  for  his  concep- 
tion is  the  fact  that  the  chromosomes  grow  and  can  thus  be  said  to  have 
a  youthful  and  an  adult  condition  and  that  they  reproduce  by  division. 
These  are  interesting  analogies  with  the  cell  itself;  but  the  cytoplasm 
as  a  mass  also  grows  from  the  size  it  has  in  the  daughter  cell  to  its  size 
in  the  adult  cell,  and  it  is  reproduced  by  division ;  still,  it  adds  nothing 
to  our  understanding  of  the  cytoplasm  to  call  it  an  individual  organism 
in  the  sense  in  which  we  so  characterize  the  cell.  Reproduction  by 
division  and  growth  are  necessary  characteristics  of  any  even  relatively 
permanent  portions  of  cells  which  assimilate,  grow,  and  divide.  It  is 
to  be  remembered,  further,  that  the  permanency  of  the  chromosome 
means  only  the  continuity  of  a  structure  which  is  undergoing  continu- 
ally its  own  series  of  cyclic  changes  in  resting-stage,  mitosis,  fusion,  etc. 
It  is  doubtless  susceptible  to  minor  alterations  due  to  its  changing  envi- 
ronment, and  is  an  active  seat  of  metabolic  changes.  And  further,  as 
to  the  significance  of  such  external  features  as  permanence  in  number, 
size,  form,  and  position  in  the  nucleus  for  the  functions  of  the  chromo- 
somes in  determining,  through  heredity,  the  structure  and  functions  of 
cells  and  cell  colonies,  we  have  as  yet  little  positive  evidence.  It  is  not 
impossible  that  the  organization  of  the  chromatin  is  a  matter  of  molecu- 
lar rather  than  a  grosser  structure.  The  doctrine  of  permanence  of 
the  chromosomes  as  structures  of  the  cell  does  not  necessarily  carry 
with  it  the  assumption  that  the  chromosomes  are  themselves  composed 
of  such  differentiated  structures,  as  is  the  cell. 

The  evidence  summarized  by  Boveri,  while  it  is  entirely  convincing 
as  to  the  permanence  of  the  chromosomes  in  the  resting  condition,  is 
almost  wholly  inferential  and  based  on  their  appearance  in  constant 
number,  form,  size,  etc.,  in  the  division  stages.  Rosenberg  (81)  has 
recently  brought  very  interesting  direct  evidence  that  the  chromosomes 
are  present  as  definitely  differentiated  structures  in  many  nuclei  in  the 
resting  condition.  He  finds  that  in  the  resting  nuclei  of  Capsella,  Zos- 
tera,  Calendula,  and  other  plants  the  resting  nuclei  show  a  series  of 
sharply  differentiated  masses  of  the  same  number  as  the  chromosome 
number  for  the  species — 32  for  Capsella,  12  for  Zostera,  32  for  Calen- 
dula— and  represent  a  form  in  which  the  chromosomes  persist  from  one 


PERMANENCE   OF   THE    CHROMOSOMES.  59 

nuclear  division  to  another.  Rosenberg  says  nothing  as  to  how  the 
chromosomes  become  connected  with  the  spindle  fibers. 

All  the  earlier  evidence  that  the  chromosomes  are  permanent  struc- 
tures in  the  cells  of  the  higher  plants  and  animals,  except  that  of  Rabl 
and  Flemming,  has  been  developed  from  a  study  of  the  form,  number, 
appearance,  etc.,  of  the  chromosomes  themselves  as  they  recur  in  each 
dividing  stage,  and  does  not  rest  on  any  proof  of  the  structural  relations 
in  the  nucleus  by  means  of  which,  in  all  the  manifold  changes  of  fusion 
and  division,  such  permanence  is  assured. 

The  nature  of  the  mechanism  by  which  the  scattered  chromatin 
elements  of  the  so-called  resting  condition  are  brought  back  into  the 
definite  form  and  positions  which  the  chromosomes  occupy  in  the  karyo- 
kinetic  figures  has  been  left  undetermined.  Boveri  (10,  n)  puts  the 
question  as  to  the  possible  nature  of  these  structures  clearly,  but  is  com- 
pelled to  resort  to  numerous  accessory  hypotheses  in  order  to  account 
for  the  regularity  with  which  the  chromosomes  reappear  in  the  same 
positions  and  number  after  their  apparent  total  disintegration  in  the 
resting  reticulum  and  the  certainty  with  which  each  daughter  chromo- 
some is  found  attached  to  one,  and  only  one,  of  the  poles  of  the  spindle. 
He  assumes  a  peculiar  affinity  on  the  part  of  each  daughter  element  by 
which  it  is  predetermined  that  the  fibers  from  a  particular  pole  will 
become  attached  to  it,  and,  further,  that  there  is  an  especial  handle 
(Henkel)  on  each  daughter  chromosome,  by  which  alone  the  fibers  may 
become  attached  to  it ;  if  a  fiber  from  one  pole  has  once  gotten  hold  of 
the  handle  on  one  daughter  chromosome,  those  from  the  other  pole 
are  excluded  from  it ;  and,  further,  other  fibers  from  the  same  pole  are 
unable  to  get  hold  of  the  handle  on  the  other  daughter  chromosome  of 
the  same  pair,  etc.  It  is  hardly  necessary  to  remark  that  the  com- 
plexity of  the  mechanism  by  which  such  a  series  of  affinities  and  capaci- 
ties could  be  brought  into  effective  action  is  well-nigh  inconceivable. 
Boveri,  in  1888  (10),  and  again  in  1897  (n),  decided  against  the  pos- 
sibility of  a  permanent  connection  between  centers  and  chromosomes 
in  favor  of  the  above  hypotheses,  and  reaffirms  his  old  position  in  his 
most  recent  contribution  on  this  subject. 

It  is  further  interesting  to  note  that  the  theory  of  the  polarity  of 
the  cell  which  has  perhaps  been  most  discussed  in  recent  years — that 
of  Heidenhain  (41) — leaves  entirely  untouched  the  question  of  the 
organization  of  the  nucleus,  and  further  assumes  not  only  that  there  is 
no  permanent  connection  of  the  central  body  with  the  nucleus,  but  that 
the  organization  of  the  cytoplasm  is  entirely  independent  of  that  of  the 


60  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

nucleus  in  the  resting  condition.  The  nucleus  lies  in  the  cytoplasm  as 
a  ball  pushed  between  the  organic  rays,  none  of  which  are  connected 
with  it.  Heidenhain  (41,  p.  504-505)  avowedly  leaves  untouched  the 
question  as  to  how  the  spindle  fibers  become  attached  to  the  chromo- 
somes, but  is  positive  that  there  is  no  fixed  relation  of  position  or  con- 
nection between  the  nucleus  and  center  in  the  resting  condition.  He 
plainly  feels  the  weakness  of  his  position  on  this  point,  and  resorts  to 
a  doubtful  analogy  in  pointing  out  that  the  discontinuity  of  nucleus  and 
center  in  the  resting  condition,  which  passes  over  into  a  continuity  in 
mitosis,  is  no  more  difficult  to  understand  than  that  independent  cells, 
i.  e.,  muscle  and  nerve,  may  become  connected  in  ontogeny. 

Heidenhain's  mechanical  theory  thus  breaks  down  at  a  critical 
point.  The  system  of  organic  rays  is  strictly  a  cytoplasmic  system, 
and  yet  the  most  important  process  in  mitosis  is  the  division  of  the 
chromosomes.  Rabl  had  both  these  factors  of  cell  organization  in 
mind,  but  his  own  observations  were  directed  most  successfully  to  the 
establishment  of  the  polarity  of  the  nucleus.  In  view  of  these  facts  it 
is  hard  to  see  the  basis  for  Heidenhain's  low  estimate  of  Rabl's  work 
(4 1,  pp.  698-702). 

Against  the  sweeping  contentions  of  Boveri  and  Heidenhain  there 
is,  none  the  less,  an  abundance  of  evidence  to  be  found  in  the  work  of 
some  of  the  best  students  of  the  animal  cell.  Meves  (63,  p.  47)  holds 
that  the  connections  between  centrosomes  and  chromosomes  by  the 
so-called  mantle  fibers  in  the  spermatocytes  of  the  salamander  are  visi- 
ble much  earlier  in  the  prophases  than  Hermann  (43)  admits,  and 
accepts  the  conception  that  the  mantle  fibers  arise  directly  from  the 
linin  network.  Kostanecki  (53,  54),  developing  still  further  the  con- 
ception of  a  system  of  organic  rays  advanced  by  Heidenhain,  holds  that 
all  the  fibrous  elements  of  the  karyokinetic  figure  are  reproduced  by 
longitudinal  division  during  mitosis,  and  regards  each  ray  and  spindle 
fiber  as  a  permanent  cell  structure. 

Conklin,  who  has  studied  the  mutual  relations  of  the  cell  structures 
more  fully  than  any  other  investigator  and  has  shown  the  relative  posi- 
tions of  nucleus,  centrosome,  sphere  substance,  etc.,  through  the  resting- 
stages  as  well  as  in  karyokinesis,  holds  (17,  p.  106)  that  it  is  evident 
that  some  kind  of  connection  exists  at  all  stages  of  the  cell  cycle  between 
the  centrosome  and  the  nucleus.  He  further  states  that  "whether 
this  connection  during  the  rest  is  in  the  form  of  fibers  (possibly  a  per- 
sistence of  those  which  previously  connected  centrosome  and  chromo- 
somes) or  is  the  expression  of  some  other  mechanical  action  or  of 
chemotropic  attraction,  does  not  appear  from  my  studies."  Conklin 


NUCLEAR    FUSION    IN    THE    ASCUS.  6l 

maintains  (17,  p.  108),  also,  that  in  the  rotations  which  occur  in  the 
telophases  the  centrosome  and  sphere  and  the  nucleus  present  their 
same  sides  to  each  other  throughout. 

Of  the  more  prominent  workers  on  the  subject  of  the  mechanism 
of  karyokinesis  who  have  obtained  positive  evidence  of  a  permanent 
connection  of  the  nuclear  content  with  the  centrosome  may  be  men- 
tioned Rabl,  Flemming,  Meves,  Kostanecki,  and  Conklin.  Those  who 
believe  that  the  contractile  fibers  each  time  form  a  new  connection  with 
the  chromosome  include  Van  Beneden,  Boveri,  Hermann,  Driiner,  and 
many  other  recent  students  of  nuclear  division  in  animal  cells. 

Montgomery  (65)  and  Paulmier  (75)  hold  that  the  connection  of 
the  spindle  fibers  with  the  chromosomes  persists  between  the  first  and 
second  maturation  divisions.  Boveri  (13)  also  accepts  this  view  and 
believes  that  thus  the  chromosome  reduction  is  effected  in  the  second 
division,  regarding  this  case  as  an  exception  to  the  general  rule.  Paul- 
mier holds  that  in  the  first  division  the  spindle  fibers  arise  by  a  special 
orientation  of  the  linin  of  the  nucleus. 

Jenkinson  (50),  who  has  approached  the  question  with  quite  differ- 
ent preconceptions  as  to  cell  structures,  and  whose  results  are  certainly 
unreliable  on  many  points,  finds  that  in  the  origin  of  the  cleavage  spindle 
of  the  axolotl  the  membrane  of  the  sperm  nucleus  appears  weakened  or 
wanting  on  the  side  where  the  centrosome  first  appears,  and  that  the 
centrosome  is  here  so  close  to  the  nucleus  as  to  appear  as  if  emerging 
from  it. 

THE    NUCLEAR    FUSION    IN    THE   ASCUS. 

The  evidence  from  the  series  of  figures  showing  the  nuclei  of  the 
ascogenous  hyphae  in  their  later  stages  of  development,  as  given  above, 
indicates  that  the  fusion  of  the  nuclei  in  the  young  ascus  does  not  result 
in  doubling  the  number  of  chromosomes  as  they  appear  in  the  succeed- 
ing divisions,  and  in  this  respect  this  nuclear  union  differs  fundament- 
ally from  any  sexual  fusion  of  nuclei  in  the  higher  plants  or  animals 
in  connection  with  which  the  chromosome  number  has  been  yet  estab- 
lished. There  is  no  visible  doubling  of  the  number  of  chromosomes  in 
the  ascus,  and  while  we  must  assume  that  the  combining  chromosomes 
maintain  their  identity,  the  centers  unite  so  intimately  that  at  least 
nothing  of  a  double  organization  is  visible.  On  the  other  hand,  the 
fusion  of  the  ascus  is  followed  at  once  by  a  synapsis  stage  and  a  triple 
instead  of  the  ordinary  double  division  of  the  spore  mother  cell.  We 
have  thus  an  immediate  apparent  reduction  of  the  number  of  the  chro- 
mosomes by  one-half,  the  16  chromatin  strands  of  the  fusing  nuclei 
appearing  as  8  strands  in  the  spirem  stage  of  the  fusion  nucleus  and 


62  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

as  8  chromosomes  in  the  equatorial  plate  a  little  later.  Doubtless  in 
the  lower  algae  and  fungi  cases  of  sexual  fusion,  followed  by  immediate 
reduction,  may  exist,  but  not  in  connection  with  any  such  elaborate 
differentiation  of  fruit-forms  as  we  find  in  the  conidia  and  ascocarps 
of  the  Ascomycetes. 

In  the  oogonium  of  the  mildew,  on  the  other  hand,  the  fusion  of 
sexual  nuclei  from  separate  gametes  is  not  followed  by  any  evidence  of 
reduction  processes,  but  results  in  a  vegetative  growth  plainly  com- 
parable to  the  development  of  a  sporophyte  generation,  as  has  so  many 
times  been  suggested  by  the  older  authors.  I  have  not  been  able  so 
far  to  count  the  chromosomes  in  the  fertilized  egg-nucleus,  but  in  the 
growth  of  the  ascogonium  and  ascogenous  hyphas  before  the  ascus  is 
formed  there  is  no  evidence  of  the  existence  of  any  synapsis  stage  or 
special  double  division,  such  as  is  now  universally  recognized  as  asso- 
ciated with  the  process  of  chromosome  reduction.  We  shall  see  also 
that  there  is  evidence  for  believing  that  the  chromosomes  of  the  nuclei 
which  fuse  in  the  ascus  are  already  bivalent  structures  as  a  result  of 
the  previous  nuclear  fusion  in  the  oogonium. 

We  must  note,  as  bearing  on  this  point,  that  while  the  process  of 
fertilization,  in  all  cases  where  it  has  been  thoroughly  investigated,  is 
the  formation  of  a  cell  with  the  double  number  of  chromosomes,  the 
combination  of  these  chromosomes  in  the  single  nucleus  may  be  either 
immediate  or  a  more  gradual  process,  as  is  conspicuous  in  the  embryos 
of  Cyclops;  and  Blackman's  (8)  and  Christman's  (15)  interesting  dis- 
coveries in  the  aecidium  of  the  rust  show  that  the  final  combination  of 
the  nuclei  may  be  delayed  through  an  indefinite  number  of  cell  genera- 
tions. The  ultimate  fusion  in  this  case  seems  to  be  associated  with  the 
process  of  chromosome  reduction  and  the  development  of  spores. 

Blackman's  discovery  of  his  so-called  vegetative  fertilization  in  the 
aecidium  of  the  rusts,  bearing  as  it  does  on  the  whole  question  of  the 
homologies  and  relationships  of  the  higher  fungi,  as  well  as  on  the 
general  question  of  fertilization  and  alternation  of  generations,  is  cer- 
tainly of  the  highest  importance.  With  his  account  and  that  of  Christ- 
man  we  may  believe  that  the  historic  question  as  to  the  sexuality  of  the 
secidium  is  finally  settled.  Blackman  finds  that  the  basal  cell  of  each 
row  of  aecidiospores  in  Phragmidium  violaceum  is  fertilized  by  the 
migration  through  its  wall  of  a  nucleus  from  a  neighboring  cell.  The 
pronuclei  do  not  fuse,  but  divide  by  conjugate  division,  and  thus  the 
cycle  of  binucleated  cells  which  terminates  with  the  nuclear  fusion  in  the 
teleutospore  is  begun.  There  is  no  wide  communicating  pore  between 
the  gametes,  as  found  by  Blackman.  The  nucleus  passes  through  the 


NUCLEAR  FUSION   IN  THE  ASCUS.  63 

wall  without  leaving  a  trace  to  indicate  its  path.  The  fertilization  is 
accomplished  by  the  nucleus  alone. 

Christman's  (15)  further  discoveries  in  the  same  line  and  on  a 
related  species  form  a  most  valuable  extension  of  Blackman's  results. 
In  Phragmidium  speciosum  Fr.  Christman  finds  an  actual  and  typical  cell 
fusion  at  the  base  of  each  row  of  ascidiospores.  The  fusion  is  between 
vertical  hyphal  cells  whose  bases  diverge  below,  indicating  that  they 
arise  from  quite  widely  separated  hyphal  branches.  The  fusing  cells 
are  equal  in  size,  and  it  is  as  an  outgrowth  of  their  combined  apices  that 
the  row  of  ascidiospores  takes  its  origin.  Here,  again,  the  nuclei  of  the 
gametes  do  not  fuse,  but  divide  simultaneously  to  form  the  pairs  of 
nuclei  found  in  the  secidiospores. 

I  shall  have  occasion  to  return  to  Blackman's  and  Christman's 
results  in  other  connections.  Here  we  are  chiefly  concerned  with  the 
fact  that  the  time  and  degree  of  the  visible  combination  of  the  sexual 
prochromosomes  is  a  variable  matter.  If  the  prochromosomes  can 
remain  either  in  one  nucleus  with  double  chromosome  number  or  in  two 
distinct  nuclei  through  part  or  all  of  the  sporophyte  generation,  it  is 
also  possible  that  they  may  combine  in  one  nucleus  into  bivalent  chro- 
mosomes and  maintain  their  identity  in  this  condition  through  the  spo- 
rophyte generation  till  a  true  reduction  occurs  in  spore  formation.  It 
is  certain  that  with  the  nuclear  organization  described  above  the  indi- 
vidual chromosomes  must  be  permanent  structures,  and  that  for  every 
chromosome  unit  which  enters  a  given  nucleus  a  corresponding  chro- 
mosome unit  must  reappear  in  the  division  of  that  nucleus. 

I  have  already  (37,  pp.  677-678)  advanced  the  view  that  the  forma- 
tion of  the  primary  nucleus  of  the  ascus  and  the  succeeding  divisions 
may  correspond  morphologically  to  the  process  of  spore  formation  at 
the  end  of  the  sporophyte  generation  in  the  ordinary  cases  of  alternation 
of  generations.  With  the  evidence  presented  above,  that  a  reduction 
of  the  number  of  chromosomes  occurs  in  the  formation  of  the  primary 
nucleus  of  the  ascus,  the  evidence  that  the  ascus,  like  the  spore  mother 
cell  of  the  moss  or  fern,  represents  the  close  of  a  sporophyte  generation 
is  apparently  strengthened;  still,  it  is  plain  that,  since  the  nuclei  that 
fuse  in  the  ascus  are  themselves  products  of  a  nuclear  fusion  in  the 
oogonium  which  must  double  the  chromosome  number,  we  must  look 
further  for  a  complete  explanation  of  the  processes  here  involved. 

I  shall  present  further  evidence  on  this  point  later ;  but  whether  we 
accept  or  reject  the  evidence  for  the  existence  of  an  alternation  of  gen- 
erations in  the  Ascomycetes  and  their  probable  congeners,  the  Florideae, 
the  problem  as  to  the  nature  of  nuclear  fusion  in  the  ascus  still  remains. 


64  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

The  problem  is  not  more  difficult,  perhaps,  in  the  case  of  the  fusion 
in  the  ascus  than  in  that  of  the  fusion  of  the  polar  nuclei  and  the  second 
sperm  nucleus  in  the  embryo-sac,  or  the  second  nuclear  fusion  described 
by  Chmielewski  ( 14)  as  taking  place  in  the  formation  of  the  zygospore 
in  Spirogyra,  or  the  fusion  vegatative  nuclei  in  endosperm  cells  (87), 
or  the  experimentally  induced  nuclear  fusions  observed  by  Nemec  (70) 
in  root  cells.  It  must  be  admitted  that  there  has  been  so  far  no  general 
agreement  as  to  either  the  morphological  or  physiological  significance 
of  any  of  these  processes,  and  doubtless  we  need  more  facts  as  to  the 
relation  of  the  nucleus  to  other  processes  in  the  cell  besides  fertilization 
before  a  final  solution  can  be  reached.  It  is  a  question,  further,  how 
much  any  two  of  the  individual  cases  mentioned  have  in  common. 
Each  may  well  be,  to  a  considerable  extent,  the  reaction  of  the  nuclei 
to  different  conditions  and  with  quite  different  results  for  the  cell. 
With  the  exception  of  the  second  fusion  in  the  zygospore  of  Spirogyra 
they  have,  however,  one  very  striking  point  in  common.  They  all  occur 
in  cells  whose  dimensions  are,  or  become,  greater  than  those  of  the  ordi- 
nary cells  with  which  they  are  associated.  This  is  strikingly  true  of  the 
ascus,  which,  as  I  have  pointed  out  above,  is  gigantic  in  size  compared 
with  any  other  cells  of  the  mildew.  Into  it  are  poured,  for  the  forma- 
tion and  nutrition  of  the  spores,  all  the  surplus  food  materials  accumu- 
lated in  the  vegetative  cycle  of  the  fungus.  The  injection  of  this 
immense  amount  of  food  material  into  the  cytoplasm  and  its  consequent 
rapid  growth  leads  naturally  to  the  expectation  that  a  correspondingly 
large  nucleus  must  be  formed ;  and,  as  I  have  pointed  out  above,  the 
nucleus  of  these  ascus  cells  is  actually  as  much  greater  than  the  ordinary 
vegetative  nuclei  as  the  ascus  is  larger  than  the  vegetative  cells. 

My  studies  of  the  nuclear  processes  in  the  ascus  have  from  the 
first  led  me  to  the  conclusion  that  the  nuclear  fusion  in  the  young  ascus 
was  correlated  in  some  way  with  the  vegetative  development  of  the  rela- 
tively gigantic  size  of  the  ascus  as  compared  with  other  cells  of  the  fun- 
gus ;  and  in  these  facts  of  relative  size  I  am  convinced  we  have  a  basis 
for  correlating  these  cases  of  nuclear  fusions  with  the  broader  facts  as 
to  the  relation  of  nuclear  dimensions  to  cell  dimensions  which  have  been 
frequently  noted  and  recently  have  been  given  striking  experimental 
demonstration  by  Gerassimoff,  R.  Hertwig,  and  Boveri.  The  fact  that 
large  cells  in  general  have  large  or  numerous  nuclei  and  that  small  cells 
have  small  or  few  nuclei  is  well  known,  but  that  this  relation  is  funda- 
mental and  necessary  was  first  shown  by  the  experimenters  just  named. 

We  may  note,  first,  Gerassimoff's  (26-29)  results.  As  is  well 
known,  by  ingenious  experimental  methods — cooling  while  cell  division 


NUCLEAR  FUSION   IN  THE  ASCUS.  65 

is  going  on,  etc. — Gerassimoff  has  been  able  to  check  the  normal  course 
of  cell  division  in  filaments  of  Spirogyra  and  to  produce  binucleated 
cells  or  to  cause  the  fusion  of  the  two  daughter  nuclei  into  a  single  pro- 
portionately larger  nucleus.  In  either  case  the  result  is  the  same.  The 
cells  with  the  larger  nuclear  mass  increase  in  size  beyond  the  norm  for 
the  species  to  which  they  belong.  These  enlarged  cells  divide,  and 
thus  filaments  are  formed  whose  cells  are  all  above  the  normal  in  size. 
In  the  binucleated  cells  with  two  fully  developed  nuclei  the  latter  tend 
ordinarily  to  repel  each  other  (29,  p.  73)  to  the  extent  necessary  to  main- 
tain their  mutually  symmetrical  position  in  the  central  region  of  the  cell. 
Still,  if  the  cells  are  weakened  or  starving,  the  nuclei  may  approach 
each  other  and  apparently  tend  to  combine.  Gerassimoff  concludes 
(29,  p.  77)  that  the  cells  of  Spirogyra  possess  the  ability  to  compensate 
for  any  disturbance  of  the  quantitative  equilibrium  between  the  mass  of 
the  nucleus  and  that  of  the  remaining  components  of  the  protoplast  by 
varying  their  rate  of  cell  division.  Increase  of  nuclear  material  leads 
to  a  delay  of  cell  division  and  a  relative  diminution  of  nuclear  material 
in  the  daughter  cells.  On  the  other  hand,  lack  of  nuclear  material  is 
followed  by  increased  frequency  in  cell  division.  It  should  be  remem- 
bered that  other  methods  might  lead  to  the  same  result,  and  if  the  main- 
tenance of  an  equilibrium  between  nuclear  and  cytoplasmic  masses  is 
a  fundamental  necessity  for  the  cell,  it  may  be  expected  that  in  different 
cases  different  means  adapted  to  the  special  conditions  of  the  individual 
cells  or  organisms  will  be  found  in  operation. 

R.  Hertwig  (44,  45)  has  reached  results  similar  to  those  of  Geras- 
simoff in  his  experiments  with  cultures  of  certain  Protozoans  (Actino- 
sphaerium,  Dileptas) .  He  finds  that  lack  of  food  leads  to  a  reduction 
of  the  volume  of  the  cytoplasm;  the  nuclei  shrink  and  a  certain  pro- 
portion of  them  actually  disintegrate.  The  nuclei  of  Actinosphaerium 
may  thus  be  reduced  from  several  hundreds  to  one  or  two.  In  overfed 
individuals  the  reverse  is  true.  It  is  thus  shown  that  the  regulative 
function  is  a  reversible  one.  Experimentally  achieved  increase  of  the 
nuclear  mass  in  Spirogyra  leads  to  the  enlargement  of  the  cytoplasmic 
mass.  Reduction  of  the  cytoplasmic  mass  by  starvation  in  Actino- 
sphaerium leads  to  a  reduction  of  the  nuclear  mass.  Boveri  (12)  has 
also  shown  by  experiments,  in  which  he  fertilized  both  nucleated  and 
non-nucleated  fragments  of  the  eggs  of  sea-urchins,  that  those  with  the 
abnormally  reduced  amount  of  nuclear  material  produce  larvae  with 
smaller  but  more  numerous  cells.  By  shaking  the  sea-urchin  eggs  just 
after  fertilization  he  was  able  also  to  achieve  the  same  result  as  did 
Gerassimoff  for  Spirogyra.  The  daughter  chromosomes  after  their 


66          SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

formation  reunite  to  form  a  single  nucleus  with  a  doubled  number  of 
chromosomes — 72  instead  of  36.  The  result  is  identical  with  that  in 
Spirogyra.  The  larva  produced  from  an  egg  so  treated  has  abnormally 
large  cells.  Boveri  further  observed  that  in  all  these  variations  the 
superficial  area  of  the  nucleus  and  not  its  cubic  content  is  proportional 
to  the  number  of  chromosomes  in  it. 

On  the  ground  of  a  general  consideration  of  the  relations  of  nuclear 
and  cytoplasmic  masses,  as  well  as  his  own,  Gerassimoff  s,  and  Boveri's 
experimental  results,  Hertwig  (45)  extends  these  conclusions  to  all 
cells  as  a  general  law  of  cell  organization,  which  he  calls  the  principle 
of  the  nucleo-cytoplasmic  relation.  Nuclear  masses  and  cytoplasmic 
masses  strive  always  to  maintain  a  definite  proportionality  in  which  they 
are  in  equilibrium.  Any  increase  in  the  mass  of  either  tends  toward 
producing  a  corresponding  increase  in  the  other;  a  reduction  in  one 
necessitates  a  reduction  in  the  other,  in  order  that  the  nucleo-cyto- 
plasmic equilibrium  may  be  maintained.  Hertwig  considers  this  rela- 
tion as  one  chiefly  of  mass,  but  it  is  plain  that  other  factors  are  also 
involved.  A  mere  equilibrium  of  mass  could  be  attained  in  the  starving 
Actinosphaeria  by  an  equal  and  proportional  reduction  of  each  nucleus, 
but  instead  of  this  the  object  is  gained  by  the  total  destruction  of  certain 
nuclei  and  the  survival  of  others.  In  Spirogyra,  also,  the  equilibrium 
in  the  binucleated  cells  at  least  might  be  established  by  the  later  com- 
pletion of  the  cell  division  which  was  artificially  interrupted.  It  is 
plain,  then,  that  factors  are  present  in  the  process  which  tend  not  only 
to  establish  a  definite  nucleo-cytoplasmic  relation  of  mass,  but  which 
also  determine  the  method  by  which  this  condition  of  equilibrium  is 
brought  about.  Equilibrium  in  the  nucleo-cytoplasmic  relation  in  the 
case  of  enlarged  cells  may  be  brought  about  either  by  the  presence  of 
two  or  more  small  nuclei  or  by  the  formation  of  a  single  nucleus  of 
proportionally  greater  size. 

If  we  now  compare  the  cell-reactions  experimentally  discovered 
by  Hertwig  and  Gerassimoff  with  those  prevailing  in  the  normal  devel- 
opment of  the  ascus,  we  shall  find  a  striking  similarity  in  all  character- 
istic features.  We  may  consider  first  the  ascus  of  the  mildew. 

The  ascus  is  to  be  developed  as  a  relatively  large  cell  to  serve  as 
a  storehouse,  with  an  abundant  supply  of  material  for  the  formation 
of  ascospores ;  and  in  order  that  the  nucleo-cytoplasmic  equilibrium  may 
be  maintained,  it  must  be  provided  with  an  excess  of  nuclear  material 
as  compared  with  the  other  cells  of  the  ascogenous  hyphge  and  the  asco- 
gonium.  There  are  several  stages  in  this  differentiation  of  the  ascus 
as  to  its  nuclear  content.  It  is  binucleated  from  the  first,  while  the 


NUCLEAR  FUSION   IN  THE  ASCUS.  67 

other  cells  mentioned  are  uninucleated ;  and,  further,  its  two  nuclei  fuse 
with  the  union  of  all  their  corresponding  parts  to  form  a  single  larger 
nucleus,  which  in  turn  grows  with  the  further  growth  of  the  ascus. 

The  binucleated  condition  of  the  young  ascus,  we  may  conceive, 
is  due  to  an  inhibition  of  cell  division,  due  in  turn,  perhaps,  to  a  cul- 
mination in  the  process  of  extra  feeding  of  the  ascogenous  cells,  which 
the  whole  structure  and  development  of  the  ascocarp  is  calculated  to 
bring  about.  Cell  division  and  nuclear  division  are  quite  independent 
processes  in  the  development  of  the  ascogonium  and  ascogenous  hyphse, 
as  we  have  seen  above.  For  a  time  the  ascogonium  and  the  ascogenous 
hyphae,  in  their  rapid  growth,  are  multi-nucleated,  but  in  the  end  cell 
division  overtakes  nuclear  division  and  the  whole  system  comes  to  con- 
sist of  uninucleated  cells,  except  the  cells  which  are  to  become  asci. 
The  binucleated  condition  remains  in  them  simply  because  cell  division 
is  inhibited  at  just  this  stage  of  development.  It  seems  probable  that 
these  ascogenous  cells  are  differentiated  as  such  simply  on  the  basis  of 
their  more  favorable  position  for  nutrition,  and  that  this  excessive  nutri- 
tion is  the  stimulus  which  inhibits  cell  division. 

It  is  a  frequently  expressed  conception  that  the  stimulus  to  cell 
division  is  given  in  a  certain  maximal  size  of  the  cell,  which,  when  it  is 
attained  by  the  growth  of  the  daughter  cells,  results  in  certain  tensions 
which  set  in  operation  the  mechanism  of  karyokinesis.  Shaper  (85)  has 
pointed  out  that  increased  volume  results  in  a  relative  diminution  of  the 
surface  area  as  compared  with  the  mass  of  the  cell,  and  since  all  nutrition 
comes  through  the  surface,  a  stage  will  be  reached  when  assimilation 
and  dissimilation  will  balance  each  other  and  growth  will  cease.  Cell 
division  now  occurs,  and  by  the  formation  of  two  smaller  daughter  cells 
a  relation  of  volume  and  surface  area  favorable  for  growth  will  again 
be  established.  Considering  the  growth  of  the  animal  egg,  Lubosch 
(58)  points  out  that  the  special  provisions  for  its  nutrition,  nurse  cells, 
etc.,  may  have  the  effect  of  inhibiting  cell  division  by  furnishing  such 
a  rich  food  supply  that  the  relative  diminution  of  absorbing  surface  will 
be  more  than  counterbalanced  and  the  cell  may  continue  to  grow  with- 
out dividing  as  long  as  the  excessive  food  supply  is  available.  The  case 
of  the  ascus  is  similar,  and  it  seems  entirely  reasonable  to  assume  that 
the  excessive  food  supply  prevents  the  separation  of  the  two  nuclei  in 
the  young  ascus  by  the  formation  of  a  cell  wall.  The  relative  excess 
of  nuclear  material  thus  accumulated  favors  the  further  growth  of  the 
cytoplasm  independently  of  the  rate  of  food  supply,  and  thus  we  get 
further  rapid  increase  in  size  of  the  ascus  cell.  We  have  thus,  in  the 
formation  of  the  ascus,  a  definite  change  in  the  habit  of  growth  of  the 


68  SEXUAL    REPRODUCTION     IN    CERTAIN     MILDEWS. 

ascogenous  cell.  While  in  the  growth  of  the  vegetative  hyphse,  the 
formation  of  sexual  cells  and  the  development  of  the  ascogonium  and 
ascogenous  hyphse  nuclear  division  are  followed  by  cell  division,  so  that 
uninucleated  cells  are  formed,  we  have  here  a  stage  in  which  cell  division 
does  not  occur  between  the  two  nuclei  of  certain  cells,  and  the  two 
nuclei  remaining  in  the  same  cell  mass  fuse  into  one.  Gerassimoff  (26) 
induced  the  interruption  of  cell  division  by  suitably  regulated  inhibitive 
stimuli  (chilling,  anesthetizing).  The  inhibition  is  self -induced  in  the 
mildew  by  the  operation  of  factors  which  are  directed  to  the  production 
of  a  large  cell  with  abundance  of  material  for  the  formation  of  spores. 

But  whatever  the  factor  or  factors  may  be  which  inhibit  cell  division 
and  thus  leave  the  young  ascus  with  two  nuclei,  this  condition  results 
exactly  as  in  Gerassimoff's  binucleated  Spirogyra  cells  and  Boveri's 
sea-urchin  eggs  with  the  double  number  of  chromosomes.  The  rela- 
tive excess  of  nuclear  material  facilitates  the  rapid  growth  of  the  ascus 
in  size.  The  cells  of  Spirogyra  do  not  complete  the  interrupted  division 
and  thus  reestablish  the  nucleo-cytoplasmic  equilibrium,  but  grow  larger, 
not  by  pathological  hypertrophy  leading  to  death,  but  in  a  normal  fashion 
which  permits  of  their  indefinite  further  division  and  growth.  The 
ascus  shows  exactly  the  same  physiological  reaction  to  its  increased 
nuclear  content.  It  immediately  grows  to  a  far  greater  mass  than  that 
of  any  of  the  other  cells  of  the  ascocarp  or  mycelium.  The  evidence 
is  thus  very  strong  that  the  doubling  of  the  nuclear  mass  in  the  young 
ascus  is  merely  a  preliminary  to  that  growth  of  the  ascus  which  is  neces- 
sary for  its  functioning  as  a  spore-sac.  The  whole  process  is  thus  placed 
in  the  category  of  nucleo-cytoplasmic  regulations  which  are  concerned 
with  maintaining  an  equilibrium  between  the  factors  of  assimilation  and 
division  in  the  cell. 

It  is  doubtless  true  that  nucleo-cytoplasmic  equilibrium  is  achieved 
many  times  by  an  increased  number  of  nuclei  in  the  cell  without  their 
fusion,  especially  in  the  algse  and  fungi.  This  is  plainly  the  case  in  the 
multinucleated  perithecial  cells  of  the  mildews  themselves  and  in  the 
multinucleated  hyphas  and  reproductive  cells  of  Pyronema  and  Asco- 
bolus.  In  both  of  these  latter  cases  increased  size  as  well  as  increased 
number  of  the  nuclei  especially  characterize  the  large  oogonia  and  the 
ascogonia.  In  the  multinucleated  endosperm  cells  and  in  Nemec's  (70) 
binucleated  root  cells  fusion  may  occur  at  once  or  the  nuclei  may  remain 
independent  and  divide  again,  whereupon  nuclear  fusion  may  occur.  In 
Gerassimoff's  (29)  experiments  fusion  of  the  nuclei  might  or  might  not 
occur.  Hence  it  seems  to  be  a  matter  of  relative  indifference,  which 


NUCLEAR  FUSION   IN  THE  ASCUS.  69 

may  be  determined  by  minor  factors  in  each  case,  whether  the  nucleo- 
cytoplasmic  equilibrium  be  established  with  or  without  nuclear  fusion. 

Nemec  (70)  has  pointed  out  that  the  fusion  of  nuclei,  far  from 
always  having  a  sexual  significance,  may  well  be  considered  in  many 
cases  merely  as  a  consequence  of  the  inclusion  of  two  nuclei  in  the 
same  cytoplasmic  system,  and  that  the  maintenance  of  many  nuclei  in 
the  large  vegetative  cells  of  the  algae  and  fungi  may  be  for  the  purpose 
of  distributing  the  nuclear  material  through  the  cell,  so  that  its  relation 
to  growth  and  metabolism  may  be  more  perfectly  and  readily  main- 
tained— a  view  which  appears  to  be  generally  accepted  by  the  students 
of  coenocytic  cells.  On  the  basis  of  his  own  experiments  he  further 
contends  that  the  cell  fusion  and  not  the  nuclear  fusion  is  the  essential 
feature  in  sexual  reproduction,  and  that  the  fusion  of  the  pronuclei  also 
may  be  regarded  as  in  the  nature-  of  a  necessary  sequence  of  the  cell 
fusion  without  thereby  detracting  in  any  degree  from  the  important 
physiological  significance  of  the  former.  Blackman's  (8)  and  Christ- 
man's  (15)  discoveries  in  the  aecidium,  discussed  above,  support  this 
view  unequivocally.  In  the  light  of  these  results  we  are  bound  to  con- 
clude that  in  the  rusts  the  process  of  nuclear  fusion  is  associated  with 
the  process  of  chromosome  reduction  rather  than  with  fertilization. 

Nemec  has  further  observed  (70)  that  these  non-sexual  fusions  in  the 
root  cells  result  in  doubling  the  chromosome  number,  which,  however, 
is  later  apparently  reduced  again  to  the  normal  number  for  the  sporo- 
phyte.  This  reduction,  he  concludes,  is  an  autoregulative  function. 
In  the  ascus  the  nuclear  fusion,  as  I  have  described  above,  results  in  an 
immediate  apparent  reduction  of  the  chromosome  number.  That  the 
reduction  is  immediate  in  the  ascus  and  occurs  somewhat  less  promptly 
in  the  root  cells  may  well  be  due  to  the  fact  that  the  whole  process  in 
the  latter  demands  new  adjustments,  while  in  the  ascus  it  is  normally 
recurrent  at  a  definite  point  in  the  life  cycle  and  may  well  have  been 
perfected  in  its  operation  by  selection. 

The  nucleus  of  the  ascus  under  normal  conditions,  since,  as  we 
shall  see  further  on,  it  presumptively  must  contain  quadrivalent  chromo- 
somes without  thereby  having  its  own  apparent  number  increased,  must 
be  considered  to  be  in  a  sense  hypertrophied  as  to  its  chromatin  content 
when  compared  with  the  ordinary  nuclei  of  the  mildew.  As  noted  also, 
the  whole  fungus  pours  its  excess  of  nutriment  into  the  ascus,  and  both 
nucleus  and  cytoplasm  increase  greatly  in  size.  The  condition  is  pos- 
sibly parallel  to  that  of  the  nuclei  in  Hertwig's  (44)  cultures  of  Actinos- 
phaerium,  which  he  kept  for  long  periods  under  conditions  of  over- 
feeding. In  such  cases  he  was  able  to  observe  that  the  hypertrophied 


/O  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

nuclei  underwent  changes  which  resulted  in  part  of  their  content  being 
thrown  out  into  the  cytoplasm  as  brown  pigment  granules.  It  is  inter- 
esting to  note  in  this  connection  that  the  abundant  highly  stainable  gran- 
ules which  I  have  described  as  present  in  the  ascus  of  Ascobolus  and 
Peziza  (35,  p.  71),  both  around  the  nuclei  and  scattered  in  the  cyto- 
plasm, originate,  according  to  the  recent  interesting  investigations  of 
Guilliermond  (34),  in  the  neighborhood  of  the  nucleus.  Guilliermond 
does  not  believe  that  the  granules  arise  directly  from  the  nucleus,  but 
thinks  it  may  play  an  indirect  role  in  their  secretion.  It  is  not  impos- 
sible that  the  formation  of  these  granules  in  the  ascus  may  be  closely 
related  to  the  formation  of  pigment  granules  described  by  Hertwig. 
Ikeno  (47)  has  described  a  throwing  out  of  chromatin  material  in  the 
case  of  the  nucleus  of  the  ascus  of  Taphrina,  which  may  be  of  a  similar 
nature. 

In  the  further  development  of  the  ascus  we  are  confronted  with 
the  peculiar  fact  that  nuclear  growth  continues  and  nuclear  division  is 
inhibited  from  a  period  prior  to  nuclear  fusion  in  the  young  ascus  till 
the  latter  is  mature  and  ready  for  the  formation  of  spores.  This  con- 
dition is  in  sharp  contrast  with  the  fact  that  in  all  the  vegetative  devel- 
opment of  the  mildew  and  the  development  of  the  ascocarp,  up  to  the 
formation  of  the  young  ascus,  nuclear  division  has  always  recurred  at 
intervals  such  as  would  prevent  the  growth  of  any  single  nucleus  beyond 
the  normal  size  for  either  vegetative  or  reproductive  cells.  We  have 
concluded  above  that  the  rich  nutrition  of  the  ascogenous  hyphse  inhibits 
cell  division  and  leads  to  the  formation  of  the  young  asci  with  two 
nuclei.  This  condition  makes  possible  a  considerable  growth  of  the 
ascus  before  the  condition  of  nucleo-cytoplasmic  equilibrium  is  reached. 
Soon,  however,  the  nuclei  continue  their  growth  in  size,  and  this  process 
continues,  as  noted,  through  the  process  of  fusion  and  after  it  till  the 
ascus  has  reached  practically  its  mature  size.  The  nucleo-cytoplasmic 
relation  is  thus  maintained  by  the  development  of  a  single  large  nucleus 
rather  than  by  the  formation  of  many  smaller  ones,  as  is  elsewhere  so 
.commonly  the  case  in  the  fungi. 

If  we  seek,  now,  the  cause  of  this  relatively  long  inhibition  of 
nuclear  division,  we  may  note  the  interesting  fact  that  the  inhibition 
lasts  only  until  the  ascus  has  reached  its  maximum  size,  when  we  may 
conclude  that  the  rich  supply  of  food  which  has  been  poured  into  it 
from  the  mycelium  begins  to  diminish.  With  this  check  in  assimilative 
processes,  reproductive  activity  is  at  once  reinaugurated  and  the  ascus- 
nucleus  divides  three  times  in  rapid  succession,  cell  division  follows, 
and  the  ascospores  are  formed.  It  seems  justifiable  to  conclude  that 


NUCLEAR  FUSION   IN  THE  ASCUS.  71 

the  excessive  assimilative  activity  in  the  ascus  has  inhibited  nuclear 
division  just  as  in  its  earlier  stages  excess  of  nutrition  inhibited  cell 
division  to  the  extent  of  leaving  the  young  asci  with  two  nuclei. 

In  the  process  of  spore  formation  we  have  again  a  most  striking 
example  of  the  controlling  influence  of  the  so-called  nucleo-cytoplasmic 
relation.  The  nucleus  of  the  ascus  divides  to  form  two  daughter  nuclei, 
and  these  in  turn  divide  successively  to  form  eight  nuclei;  but  in 
thus  passing  from  the  uninucleated  to  the  multinucleated  condition  the 
nucleo-cytoplasmic  equilibrium  is  maintained.  The  two  daughter  nuclei 
are  proportionally  smaller  than  the  mother  nucleus,  and  the  four  and 
eight  nuclei  in  the  end  bear  approximately  the  same  relation  to  their 
cytoplasmic  masses  as  did  the  primary  nucleus  of  the  ascus  to  the  cyto- 
plasm of  the  entire  ascus.  The  two  nuclei  which  become  the  centers 
for  the  formation  of  spores  grow  to  a  somewhat  larger  size  than  the 
remaining  six,  and  accordingly  the  mass  of  cytoplasm  included  in  the 
two  spores  is  more  than  one-fourth  of  that  in  the  entire  ascus. 

A  careful  study  of  the  processes  involved  in  the  development  of  the 
ascus  leads  thus  to  the  conclusion  that  it  is  in  its  nature  as  a  spore- 
producing  organ  that  we  find  the  explanation  of  the  various  nuclear  and 
growth  phenomena  which  characterize  it.  By  inhibition  of  cell  division 
at  a  certain  stage  in  the  development  of  the  ascogenous  hyphse  the  ascus 
is  formed  as  a  binucleated  cell,  and  the  excess  of  its  nuclear  content 
makes  possible  a  proportionate  development  of  its  cytoplasm.  Its  nuclei 
grow  and  fuse  and  nuclear  division  is  further  inhibited,  and  thus  the 
relatively  enormous  size  of  the  uninucleated  ascus  cell  is  atained.  With 
the  diminution  of  food  supply  nuclear  and  cell  division  are  resumed  and 
the  uninucleated  ascospores  are  formed,  in  which,  again,  the  nucleo- 
cytoplasmic  relation  is  also  maintained. 

A  comparison  of  the  processes  thus  described  and  analyzed  with 
those  associated  with  fertilization  elsewhere  makes  it  still  clearer  that 
the  development  of  the  ascus  can  not  in  any  sense  be  compared  with 
that  of  the  egg.  Much  attention  has  been  devoted  to  the  problem  of 
the  conditions  which  lead  to  the  formation  of  the  immense  yolk  masses 
of  some  animal  eggs  and  the  relation  of  yolk  formation  to  the  size  of 
the  germinal  vesicle.  There  is  no  question  that  we  have  here  an  illus- 
tration of  the  principle  of  the  nucleo-cytoplasmic  relation,  but  in  every 
case  the  growth  of  the  yolk  mass  is  a  purely  vegetative  process  and  is 
the  preparation  for  fertilization.  In  the  ascus  nuclear  fusion  is  followed 
by  inordinate  growth  in  the  mass  of  the  cell.  (Compare  figs.  37  to  39, 
which  are  magnified  2250,  with  figs.  53  and  54,  which  are  magnified 
1500.)  With  the  possible  exception  of  a  few  of  the  algae,  in  which 


72  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

the  nuclear  phenomena  are  yet  to  be  determined  with  certainty,  the 
union  of  the  male  and  the  female  pronuclei  is  nowhere  followed  by  such 
a  growth.  Fertilization  is  the  signal  for  rapid  nuclear  division,  fol- 
lowed at  once  or  later  by  cell  division,  except  in  cases  where  the  fertil- 
ized egg  is  to  serve  as  a  resting  stage.  Dangeard's  own  theory  of  sex- 
uality, based  on  the  character  of  the  gametes  in  the  lower  green  algae, 
is  entirely  opposed  to  the  assumption  that  the  fusion  in  the  developing 
ascus  is  equivalent  to  an  ordinary  union  of  sexual  pronuclei.  On  the 
other  hand,  the  growth  phenomena  of  the  ascus  are  just  what  would 
be  expected  in  a  highly  specialized  spore  mother  cell. 

It  is  possible  that  the  nuclear  fusion  in  the  ascus,  arising  wholly 
as  I  have  described  above  in  connection  with  the  maintenance  of  the 
nucleo-cytoplasmic  equilibrium  in  the  large  ascus  cell,  may  still  func- 
tionally satisfy  in  some  minor  degree  the  requirements  of  a  sexual 
fusion  in  case,  in  the  course  of  development,  it  should  be  brought  about 
that  the  nuclei  which  so  combine  arise  from  a  widely  separated  nuclear 
ancestry.  I  have  discussed  this  possibility  in  connection  with  both  the 
Basidiomycetes  and  the  rusts  in  former  papers  (400  and  46),  and  have 
pointed  out  that  it  is  possible,  though  not  at  all  proven,  that  the  fusions 
in  the  basidia  and  teleutospores  may  in  some  degree  have  functionally 
replaced  a  true  sexual  union  of  nuclei,  though  occurring  at  the  close  of 
a  sporophyte  generation.  This,  of  course,  does  not  mean  that  in  any 
sense  whatever  basidia  or  teleutospores  can  be  considered  as  morpho- 
logically equivalent  to  oogonia,  nor  that  the  series  of  cells  from  which 
they  arise  are  morphologically  a  gametophore,  as  Dangeard  maintains. 
Blackman's  and  Christman's  discoveries  show  beyond  all  question  that 
the  morphological  equivalents  of  the  oogonia  and  antheridia  of  other 
related  fungi  and  algae  are  not  the  teleutospores  formed  at  the  end  of 
a  long  series  of  binucleated  cells.  In  the  conjugating  cells  at  the  base 
of  the  rows  of  aecidiospores  we  find  the  real  equivalents  of  the  sexual 
cells  elsewhere.  Blackman's  fertile  cell  is  doubtless  a  modified  egg  cell. 
The  other  cell  of  the  pair  we  must  conclude  is  a  new  structure  which 
has  taken  on  a  sexual  function,  since  the  spermatia,  which  are  doubtless 
the  equivalents  of  the  male  cells  of  other  groups,  are  still  in  existence  in 
the  rusts.  This  carries  with  it  the  further  conclusion  that  the  processes 
in  the  teleutospore  and  basidium  form  a  parallel  to  those  found  in  the 
spore  mother  cells  of  higher  plants. 

Blackman  on- this  basis  denies  all  sexual  significance  to  the  fusion 
in  the  teleutospore  and  considers  the  binucleated  cells  of  the  uredo  and 
teleuto  mycelia  as  entirely  equivalent  physiologically,  as  well  as  mor- 
phologically, to  the  uninucleated  cells  of  the  sporophyte  of  the  higher 


NUCLEAR  FUSION    IN   THE  ASCUS.  73 

plants,  since  both  contain  the  double  number  of  chromosomes.  While 
this  is  possible,  I  doubt  if  the  evidence  proves  quite  so  much.  The 
tendency  certainly  exists  among  the  most  recent  students  of  reducing 
phenomena,  accepting  the  suggestions  of  De  Vries  (96)  and  still  earlier 
evidence  from  the  side  of  the  zoologists  (65,  66),  to  assume  that  the 
entire  interchange  of  hereditary  units  or  of  hereditary  influences  between 
male  and  female  chromosomes  takes  place  in  synapsis  or  the  associated 
stages.  With  this  view  it  is  a  matter  of  indifference,  as  Blackman  main- 
tains, whether  the  nuclei  of  the  gametes  combine  into  one  early  or  late 
in  the  life  of  the  sporophyte.  One  nucleus  with  the  double  number  of 
chromosomes  is  exactly  the  equivalent  of  two  distinct  nuclei  which 
divide  by  conjugate  division.  Still,  it  is  to  be  questioned  whether  this 
is  actually  the  case  physiologically.  It  may  very  well  be  that  the  male 
and  female  chromosomes  in  the  nuclei  of  the  sporophyte  of  the  higher 
plants,  though  maintaining  their  individuality  as  permanent  structures, 
can  still  exert  chemical  or  other  influences  upon  each  other  in  some 
degree  as  a  result  of  their  close  association  in  the  same  nuclear  cavity, 
whether  or  not  an  actual  interchange  of  substance  occurs  between  them. 
The  facts  of  bud  variation,  adaptation  to  environment,  and  other  modi- 
fications occurring  during  the  life  of  the  individual  suggest  that  this 
may  be  the  case.  If  such  interchange  takes  place  in  a  fusion-nucleus 
and  does  not  occur  in  a  binucleated  cell  arising  from  conjugate  division, 
then  the  fusion  in  the  teleutospore  may  mean  more  than  the  reduction 
process  found  in  the  spore  mother  cells.  A  result  which  is  achieved 
immediately  in  the  union  of  the  nuclei  of  the  sperm  and  egg,  or  later 
during  the  association  of  the  male  and  female  chromosomes  in  the  sporo- 
phyte, may  here  be  accomplished  at  the  stage  of  chromosome  reduction. 
It  may  be  that  the  nuclear  fusion  everywhere  means  more  than  a  mere 
preliminary  adjustment  making  ready  for  reduction.  I  am  inclined  to 
think  that  this  is  the  case  and  that  the  fusion  in  the  teleutospore  may 
involve  in  some  degree  the  changes  resulting  from  sexual  union  beyond 
those  which  arise  from  such  a  cytoplasmic  fusion  as  results  in  the  binu- 
cleated cell,  as  described  by  Blackman  and  Christman. 

The  question  further  arises,  granting  that  this  difference  between 
reduction  phenomena  in  spore  mother  cells  and  the  processes  in  the 
teleutospore  exists,  whether  it  is  of  such  a  nature  as  to  replace  in  any 
degree,  when  the  nuclei  which  fuse  come  from  a  separated  ancestry, 
the  ordinary  sexual  conjugation  of  cells  and  nuclei  as  they  are  found 
elsewhere  in  algse  and  fungi.  There  seems  no  doubt,  in  view  of  'Miss 
Nichols's  (71)  results,  that  in  certain  Basidiomycetes  the  origin  of  the 
binucleated  cells  is  not  found  in  any  such  definite  structures  or  at  any 


74  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

such  fixed  point  in  the  life  cycle  as  in  the  rusts,  and  that  in  this  respect 
the  Basidiomycetes  represent  a  further  stage  in  development  following 
the  condition  described  in  the  rusts  by  Blackman  and  Christman.  There 
are  two  possibilities  as  to  the  causes  that  have  led  to  this  further  stage 
in  development.  Either  sexual  reproduction  by  the  union  of  differen- 
tiated gamete  cells  is  gradually  disappearing  under  the  influence  of  the 
particular  habits  of  life  of  the,fungi,the  conditions  in  the  higher  Basidio- 
mycetes representing  a  further  stage  in  this  process,  or  the  new  proc- 
esses of  conjugate  division,  followed  by  nuclear  fusion,  in  connection 
with  chromosome  reduction  at  the  close  of  a  sporophyte  generation,  has 
tended  to  replace  the  earlier  sexual  fusion.  Blackman  is  of  the  opinion 
that  such  fertilizations  by  vegetative  cells,  as  he  finds  in  Phragmidium, 
will  also  be  found  in  the  higher  Basidiomycetes,  but  Miss  Nichols  has 
made  it  plain  that,  for  certain  forms  at  least,  no  such  assumption  is  at 
all  probable,  and  it  is  further  possible  that  there  are  reduced  forms 
among  the  rusts  in  which  the  same  condition  prevails. 

On  either  of  the  above  hypotheses  such  forms  are  possible,  and  if 
the  second  hypothesis  is  accepted  we  have  the  key  to  the  explanation 
of  the  phenomena  involved  in  the  two  nuclear  fusions  found  in  the 
mildews,  Pyronema,  and  presumably  other  Ascomycetes.  I  have  pre- 
sented above  the  evidence  that  nuclear  fusion  in  the  ascus  arises  in  con- 
nection with  the  processes  involved  in  the  maintenance  of  the  nucleo- 
cytoplasmic  relation  during  the  development  of  the  relatively  enormous 
size  of  the  ascus  cell,  and  have  pointed  out  that  in  its  origin  in  this 
fashion  it  has  nothing  to  do  with  sexual  reproduction  in  any  respect. 
If,  however,  we  may  assume  that,  with  the  development  of  a  separate 
ancestry  for  the  fusing  nuclei  by  simultaneous  nuclear  division,  as  found 
in  the  bent-end  cells  of  the  ascogenous  hyphae  in  Pyronema  and,  accord- 
ing to  Maire,  in  a  series  of  binucleated  ascogenous  cells  in  Galactinia 
and  Acetabula  (see  also  31),  the  process  has  gained  in  some  degree  a 
functional  equivalence  for  sexual  fusions,  we  can  further  assume  that 
this  condition,  perhaps  working  together  with  other  influences  such 
as  have  led  to  parthenogensis  in  other  fungi,  has  made  possible  the 
occurrence  of  parthenogenesis  or  even  apogamy  in  such  Ascomycetes, 
for  example,  as  Pleospora  and  Teichospora.  Such  conjugate  nuclear 
division  would  originate,  not  as  it  apparently  does  at  present  in  the 
rusts  through  the  failure  of  the  pronuclei  to  combine  in  one,  but  in 
connection  with  the  development  of  the  spore  mother  cell,  as  in  Pyro- 
nema. From  this  point  we  might  expect  the  process  to  work  back 
in  the  ascogenous  hyphae,  as  it  appears  to  be  doing  in  Galactinia  and 
Acetabula.  Ultimately  it  might  reach  the  egg-cells  and  result  in  the 


NUCLEAR  FUSION   IN  THE  ASCUS.  75 

condition  found  now  in  the  rusts.     Briefly,  the  hypothesis  involves  that 
the  development  of  conjugate  nuclear  division  and  the  maintenance  of 
separate  lines  of  nuclear  descent  in  the  ascogenous  hyphse  might  tend 
to  give  to  the  nuclear  fusions  in  the  ascus  a  sexual  value  in  addition  to 
their  original  and  more  fundamental  significance  in  maintaining  the 
nucleo-cytoplasmic  relation  in  the  enlarged  ascus  cell,  and  that  thus,  in 
turn,  parthenogenesis,  and  later  even  apogamy,  may  have  resulted  in 
forms  in  which  conjugate  nuclear  division  in  the  ascogenous  hyphse 
had  become  established.     Such  an  hypothesis  as  to  the  possible  dis- 
appearance of  the  fusion  of  sexual  gametes  and  its  replacement  by 
the  independent  fusion  in  the  ascus  of  nuclei  of  separated  ancestry 
carries  with  it  no  implication  that  the  ascus  has  become  the  morpho- 
logical equivalent  of  an  oogonium.     The  fusion  in  the  ascus  would  be 
only  analogous  to  and  not  homologous  with  a  true  sexual  fusion,  nor 
would  this  hypothesis  affect  in  any  way  our  conception  of  the  mor- 
phology of  the  ascocarp.     The  ascus  is  a  new  structure  which  originated 
as  an  outgrowth  of  an  ascogonium,  which  in  turn  was  produced  by  the 
germination  of  a  fertilized  egg.     The  two  fusions  must  be  expected  to 
coexist  for  a  time  in  the  same  life  cycle,  as  is  actually  the  case  in  all 
Ascomycetes  whose  nuclear  history  is  fully  known. 

As  I  have  pointed  out  before,  it  still  remains  to  determine  how 
many  such  cases  there  are  among  the  Ascomycetes  and  whether  the 
genera  mentioned  are  really  apogamous  or  parthenogenetic  or,  as 
Blackman's  discovery  suggests,  whether  they  may  not  possess  a  fertiliza- 
tion by  the  migration  of  vegetative  nuclei  in  their  initial  cells.  It  is 
certainly  most  highly  desirable  that  we  should  have  a  full  account  of 
the  development  of  the  ascogenous  hyphae  and  the  behavior  of  the 
nuclei  in  some  such  type.  In  the  case  of  the  forms  referred  to  above 
also,  in  which  Maire  (59,  60)  has  reported  that  the  asci  arise  from  a 
series  of  binucleated  cells,  this  series  in  one  case  taking  its  origin  in  a 
recurved  hyphal  tip  such  as  usually  gives  rise  directly  to  an  ascus,  H 
is  of  the  highest  importance  to  know  how  the  ascocarps  originate. 
Until  this  is  settled  it  is  difficult  to  judge  with  any  certainty  of  the 
significance  of  Maire's  observations.  It  is  to  be  hoped  that  in  the  near 
future  we  may  have  a  full  account  of  the  nuclear  phenomena  in  the 
development  of  the  ascocarp  of  some  parthenogenetic  or  apogamous 
form.  Unfortunately,  the  forms  which  have  so  far  been  reported  as 
developing  their  ascocarps  in  some  other  way  than  from  a  sexual  appa- 
ratus have  not  shown  themselves  favorable  for  the  investigation  c 
nuclear  phenomena. 


76  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

RELATIONS    OF    ASCOMYCETES    AND    BASIDIOMYCETES. 

Maire's  (62)  discovery  of  regularly  binucleated  cells  in  the  carpo- 
phore of  the  Basidiomycetes,  confirmed  by  myself  (400)  and  others,  has 
firmly  established  the  conception  of  a  phylogenetic  relationship  between 
this  group  and  the  rusts.  The  inferences  drawn  by  older  authorities 
from  the  resemblance  of  the  promycelium  of  the  rusts  to  the  basidium 
of  Auricularia  are  thus  confirmed.  Maire  accepts  the  conception  of  an 
alternation  of  generations  in  the  Basidiomycetes  and  holds  that  the 
union  of  nuclei  in  the  basidium  involves  the  reduction  process  found 
in  the  spore-mother-cell  stage  of  the  higher  plants,  thus  rejecting  spe- 
cifically Dangeard's  conception  of  the  basidium  as  an  oogonium. 

There  can  be  no  question,  in  view  of  the  general  agreement  of 
other  authors  (15,  8,  46,  400),  that  Maire,  like  Sappin,  Trouffy,  and 
Dangeard,  has  been  misled  as  to  the  number  of  chromosomes  in  the 
rusts,  the  Ascomycetes,  and  the  Basidiomycetes  by  faulty  methods  of 
fixation  which  have  caused  the  chromosomes  to  stick  together  in  clumps. 
Guilliermond  (31,  33)  finds  the  number  of  chromosomes  in  the  divisions 
in  the  ascus  to  be  8  in  Aleuria  cerea,  12  in  Peziza  cortinus,  16  in  Peziza 
rutilans,  and  8  in  Peziza  vesiculosa.  I  have  found  8  chromosomes  in 
the  asci  of  Ascobolus  furfuraceus  and  Peziza  stevensoniana  (35),  8  in 
the  asci  of  Erysiphe  communis  (40),  10  in  Pyronema  confluens  (40), 
and  8  in  Phyllactinia.  In  the  face  of  these  facts  it  is  difficult  to  see  how 
Maire  and  Dangeard  can  maintain  that  there  are  probably  4  chromo- 
somes in  all  Ascomycetes.  Dangeard  originally  concluded  there  were 
8  chromosomes  in  the  conidia  of  Sphaerotheca  castagnei,  but  has  now 
changed  his  estimate  to  4,  in  harmony  with  Maire.  It  is  plain  that  no 
such  simple  conditions  exist  as  Maire  imagines  when  he  contends  for 
the  universal  occurrence  of  2  chromosomes  in  the  rusts,  4  in  the  Basidi- 
omycetes, and  4  in  the  Ascomycetes.  Maire  has  apparently  seen  more 
or  less  vaguely  the  true  chromosomes  in  the  prophases,  but  here  he  was 
not  able  to  make  out  any  constancy  in  the  number. 

Maire  (62)  holds  that  the  stage  corresponding  to  a  true  fertiliza- 
tion is  that  at  which  the  binucleated  condition  arises  in  the  hyphal  cells. 
He  was  not,  however,  able  to  determine  with  certainty  the  method  of 
origin  of  the  binucleated  condition,  nor  to  demonstrate  the  existence  of 
simultaneous  or  conjugate  nuclear  division,  so  that  he  leaves  the  initial 
point  of  the  sporophyte  generation — the  formation  of  a  fertilized  egg — 
still  uncertain  for  the  Basidiomycetes.  Miss  Nichols's  (71)  researches 
show  that  the  binucleated  condition  is  present  in  the  mycelium  of  several 
forms  and  apparently  does  not  originate  at  any  constant  stage  nor  in 
any  specially  differentiated  structures  in  the  life  cycle  of  the  fungus. 


ASCOMYCETES   AND    BASIDIOMYCETES.  77 

It  is  especially  of  interest  that  Miss  Nichols  finds  binucleated  cells  regu- 
larly present  in  the  rhizomorphs  of  a  large  number  of  widely  separated 
genera. 

The  observations  of  Miss  Nichols  and  those  of  Blackman  and 
Christman  do  not  make  it  any  easier  to  assume  a  phylogenetic  relation- 
ship between  Ascomycetes  and  Basidiomycetes.  It  is  highly  probable 
that  such  resemblances  as  exist  between  the  ascocarp  and  the  carpophore 
are  due  to  the  duplication  of  unrelated  forms  under  similar  develop- 
mental conditions,  which  occurs  at  many  other  points  in  the  plant  king- 
dom. While  a  conjugation  of  gametes  is  present  in  the  aecidium,  it  is 
still  plain,  from  the  existence  of  the  now  functionless  spermatia,  that 
the  present  sexual  fusions  are  highly  modified  processes  which  have 
returned  in  the  character  of  the  gamete  to  something  like  a  zygosporic 
type  from  what  originally  was  doubtless  a  true  carposporic  method  of 
reproduction.  There  is  no  evidence  of  any  such  modification  in  the 
sexual  apparatus  of  the  Ascomycetes,  and  in  them  also  there  is  at  most 
only  the  beginning  of  the  long  series  of  regularly  binucleated  cells  which 
characterize  the  rusts  and  Basidiomycetes.  Still,  the  fusion  in  the 
basidium  may  have  had  an  origin  similar  to  that  suggested  above  for 
the  fusion  in  the  ascus,  and,  combined  with  conjugate  division,  may 
gradually  have  led  to  the  apogamous  condition  which,  it  seems  probable, 
is  found  in  the  Basidiomycetes.  The  rusts  on  this  hypothesis  represent 
a  condition  when  conjugate  division  has  worked  back,  in  the  life  history 
of  the  sporophyte,  to  the  stage  of  fusion  of  the  gametes,  thus  replacing 
the  nuclear  fusion  in  the  egg  which  must  probably  be  assumed  to  have 
occurred  in  some  more  primitive  type  from  which  the  rusts  have  devel- 
oped. If  this  more  primitive  type  was,  as  Blackman  believes,  one  of 
the  red  algae,  we  must  probably  consider  that  two  diverging  series  of 
fungi,  the  Ascomycetes  and  the  Basidiomycetes,  had  their  origins  in 
this  group. 

As  has  been  many  times  noted,  one  of  the  commonest  grounds  for 
the  assumption  of  a  relationship  between  Ascomycetes  and  Basidio- 
mycetes and  the  relationship  of  these  groups  to  the  Floridese  lies  in  the 
generally  suggested  physiological  parallelism  between  ascocarps,  carpo- 
phores, and  cystocarps  with  each  other  and  with  the  sporogonium  of 
the  liverwort  and  moss.  A  functional  resemblance  between  the  struc- 
tures in  question  is  apparent,  and  Wolf  (99)  has  described  a  reduction 
of  the  number  of  chromosomes  in  connection  with  the  formation  of  the 
carpospores  in  Nemalion.  The  evidence,  however,  on  which  he  bases 
his  conclusion  is  not  very  convincing.  Such  comparisons  also  still 
leave  the  nature  of  the  tetraspores  and  the  occurrence  of  specialized 


78  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

tetrasporic  as  distinct  from  sexual  plants  unexplained,  and  their  validity 
is  to  be  settled  by  the  determination  of  the  chromosome  number  in  each 
case.  Mottier's  (67)  discovery  that  the  number  of  chromosomes  in  the 
first  division  in  the  tetrasporange  of  Dictyota  is  16,  which  is  about  half 
that  found  in  the  vegetative  cells  of  the  plant  which  bears  the  tetra- 
sporanges,  and  the  evidence  brought  by  Williams  (98)  that  the  tetra- 
spores  of  Dictyota  develop  into  sexual  plants,  while  the  eggs  develop 
into  tetrasporic  plants,  lead  us  to  the  conception  of  quite  a  different  set 
of  possible  homologies,  as  will  be  further  noted  below. 

Better  evidence  for  a  relationship  between  the  red  algae  and  Asco- 
mycetes  is  found  in  the  method  of  fertilization  by  a  trichogyne  as  found 
in  the  lichens,  Laboulbeniacese  and  Pyronema,  and  in  the  fact  that  in 
both  groups  the  fertilized  egg  through  its  further  development  remains 
in  organic  continuity  with  the  plant  which  bore  it,  instead  of  being  set 
free  to  begin  an  independent  existence.  It  was  on  this  ground  that 
De  Bary  proposed  the  conception  of  the  carpogonium  to  include  the 
female  cell  of  both  the  Ascomycetes  and  the  red  algae. 

Davis  (22),  in  a  critical  review  of  the  relationships  of  the  higher 
fungi  and  algae,  while  admitting  the  force  of  the  evidence  in  favor  of  a 
derivation  of  the  Ascomycetes  from  the  red  algae  through  the  Laboul- 
beniacese, is  inclined  to  the  assumption  of  a  relationship  between  the 
lower  Ascomycetes  and  the  Phycomycetes  and  to  the  belief  that  the 
Ascomycetes  may  be  a  polyphyletic  group.  Blackman  is  of  the  opinion 
that  such  a  vegetative  fertilization  as  he  finds  may  also  be  present  in  the 
higher  Basidiomycetes  and  presumably  also  in  those  rusts  which  seem 
to  lack  an  aecidium.  Further  studies  in  the  rusts  in  the  light  of  Black- 
man's  and  Christman's  discoveries  may  be  expected  to  clear  up  many 
difficult  points  as  to  relationships  among  the  higher  fungi  and  algae.  It 
is  interesting  to  note  that  Sappin-Trouffy  concluded  (84)  that  in  some 
cases  the  binucleated  condition  appears  first  in  the  teleutospore  sorus. 

Blackman  does  not  believe  that  Coleosporium  sonchi-arvensis,  as 
described  by  Holden  and  Harper  (46)  really  lacks  a  true  aecidial  stage. 
The  form  is  a  very  common  and  familiar  one  which  I  have  collected 
for  many  years  without  being  able  to  find  any  suggestion  of  an  associ- 
ation with  an  aecidial  stage  on  a  conifer,  where  one  would  naturally 
expect  it  to  occur.  It  is  my  opinion  that  this  is  a  form  with  reduced 
life  cycle,  but  I  do  not  hold  that  this  opinion  is  of  any  final  value  in  the 
absence  of  proper  culture  experiments  on  our  American  forms.  Still, 
it  is  hardly  to  be  doubted  that  such  types  do  exist,  and  it  would  make 
no  difference  with  the  conclusions  suggested  whether  the  binucleated 


ALTERNATION    OF    GENERATIONS.  79 

condition  arises  in  the  sporidium  or  later  in  the  growth  of  the  mycelium. 
Still,  no  claim  for  completeness  was  made,  since  we  did  not  germinate 
the  sporidia  nor  determine  the  nature  of  the  early  stages  of  the  mycelium 
which  arises  from  them.  As  to  the  question  of  a  name  for  the  fungus, 
in  the  absence  of  definite  evidence  it  is,  in  my  opinion,  poor  policy  to 
reject  this  commonly  used  name  (24)  until  by  culture  experiments  posi- 
tive proof  of  its  identity  and  relationships  has  been  attained.  The  main 
conclusions  reached  in  the  paper  in  question  were  that  much  more  differ- 
entiated and  normal  division  figures  were  present  in  the  rusts  than  had 
up  to  that  time  been  described,  and  especially  that  the  then  current 
conceptions  as  to  the  number  of  chromosomes  in  the  nuclei  of  the  rusts 
were  entirely  wrong.  In  both  of  these  points  Blackman's  results  con- 
firm our  own. 

If,  further,  as  Blackman  believes,  the  rusts  may  have  arisen  from 
the  Florideae,  it  is  probable  that,  at  a  time  when  the  spermatia  were  the 
functional  male  cells,  there  was  a  fusion  of  nuclei  immediately  in  the 
fertilized  cell.  If,  at  this  stage  in  the  development  of  the  group,  a 
sporophyte  generation  leading  to  the  formation  of  such  spore  mother 
cells  as  the  basidium  were  in  existence,  it  is  not  improbable  that  two 
nuclear  fusions  may  have  been  included  in  such  a  life  cycle.  On  the 
other  hand,  from  this  standpoint  alone  the  opposite  conception  that  the 
various  spore  forms  of  the  rust  arose  gradually  in  connection  with  a 
progressive  deferring  of  the  fusion  of  the  nuclei  of  the  gametes  seems 
also  plausible.  There  are,  however,  other  facts  to  be  taken  into  con- 
sideration. 

ALTERNATION    OF    GENERATIONS  IN  THE  HIGHER  FUNGI. 

It  seems  entirely  clear,  from  our  knowledge  of  the  significance  of 
the  chromosome  number  in  nuclear  division,  in  sexual  reproduction, 
and  in  the  alternation  of  generations  in  the  higher  plants,  that  the  deter- 
mination of  the  number  of  the  chromosomes  at  the  important  stages 
in  the  life  cycles  of  the  higher  fungi  will  indicate  definitely  the  true 
nature  of  the  difficult  phenomena  we  are  considering,  and  there  is  no 
doubt  that  the  problem  is  soluble  along  these  lines.  We  have  abundant 
evidence  that  in  the  asci  the  chromosomes  are  very  sharply  differentiated 
structures,  which  can  be  counted  with  certainty.  It  is  certain  that,  as 
shown  above,  the  nuclear  fusion  in  the  ascus  does  not  alter  the  apparent 
number  of  the  chromosomes  found  in  the  fusion  nucleus  and  its  off- 
spring as  compared  with  the  individual  nuclei  which  combined.  The 
fusion  of  the  nuclei  involves  the  union  of  the  chromosomes  presumably 
in  pairs,  and  thus,  through  synapsis  and  the  triple  division  which  follows, 


SO  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

a  reduction  of  the  number  of  chromosomes  is  effected.  The  discov- 
eries of  Blackman,  and  even  more  convincingly  those  of  Christman,  by 
proving  that  the  binucleated  cells  of  the  rusts  originate  in  an  unques- 
tionable fertilization,  have  put  the  existence  of  an  alternation  of  genera- 
tions in  these  forms  beyond  question.  It  is  to  be  noted  also  what  posi- 
tive support  the  conditions  in  the  rust  give  to  the  doctrine  of  the  inde- 
pendent persistence  of  the  prochromosomes  throughout  the  sporophyte 
generation,  which  was  beginning  to  be  inferred  from  the  phenomena  in 
the  higher  animals  and  plants.  When  the  nuclei  of  the  gametes  persist 
as  independent  structures  up  to  the  time  of  chromosome  reduction  in 
the  spore  mother  cell,  there  can  be  no  question  of  the  independent  per- 
sistence of  the  individual  chromosomes  from  the  two  gametes.  This 
condition  also  enables  us  to  establish  beyond  reasonable  doubt  the 
existence  of  a  stage  in  the  life  cycle  with  cells  containing  the  double 
chromosome  number  without  an  actual  counting  of  the  chromosomes. 
It  is,  of  course,  highly  desirable  that  the  number  of  chromosomes  in 
the  division  in  the  teleutospore  and  elsewhere  be  established  by  actual 
counting,  but  there  can  be  no  question,  even  without  this  further  evi- 
dence, that  the  main  conclusions  of  these  authors  are  fully  justified. 
The  binucleated  cells  of  the  rust  represent  a  stage  with  double  chromo- 
some number  arising  in  a  process  of  fertilization  and  closing  with  a 
reduction  of  the  chromosome  number.  A  parallel  with  the  alternation 
of  generations  in  the  higher  plants  is  thus  fully  established. 

If  it  could  be  further  shown  that  while  the  antheridia  and  oogonia 
unite  and  the  male  nucleus  comes  to  lie  in  the  egg  beside  the  female 
nucleus  they  still  do  not  fuse,  but  maintain  their  independent  existence 
through  the  development  of  the  ascogonium  and  ascogenous  hyphse, 
finally  combining  in  the  ascus,  we  should  have  in  the  mildews  and  Pyro- 
nema  an  apparent  parallel  to  the  nuclear  phenomena  in  the  rust.  Since, 
however,  the  cells  of  the  ascogonium  in  the  mildews  are  uninucleated 
and  there  is  no  evidence  of  any  provision  for  maintaining  separate 
and  parallel  series  of  nuclei,  such  as  are  present  in  the  rusts,  it  is  plain 
that  even  if  we  did  not  have  a  fusion  of  nuclei  in  the  oogonium  we 
could  assume  no  close  parallelism  between  the  mildews  and  rusts  on 
this  basis.  If  we  should  assume  also  that  in  Pyronema  the  male  and 
female  nuclei  only  become  mingled  but  do  not  fuse  in  the  oogonium, 
there  would  seem  to  be  equally  little  chance  for  maintaining  any  distinct 
lines  of  nuclear  descent  in  the  multinucleated  cells  of  the  ascogenous 
hyphae  until  at  the  very  close  of  their  development,  when  simultaneous 
nuclear  division  occurs  and  provision  is  thus  made  that  the  nuclei  of 
the  ascus  shall  at  least  not  be  sister  nuclei.  This  single  simultaneous 


ALTERNATION    OF    GENERATIONS.  8 1 

division  of  nuclei  can,  however,  at  the  most  be  regarded  as  no  more 
than  a  mere  beginning  as  compared  with  the  indefinite  series  of  conju- 
gate divisions  occurring  in  the  rusts. 

As  compared  with  the  rusts,  then,  we  have  in  the  mildews  and 
Pyronema  no  series  of  binucleated  cells  formed  by  conjugate  division, 
and  we  do  find  positive  evidence  that  the  sexual  nuclei  fuse  in  the 
oogonium.  Still,  I  am  convinced  that  in  the  Ascomycetes  as  well  as 
in  the  rusts  we  have  a  true  alternation  of  generations.  The  explana- 
tion of  these  differences,  combined  with  so  much  of  general  similarity 
between  Ascomycetes  and  Basidiomycetes,  is  given  in  a  further  point 
of  difference  between  the  two  groups. 

In  the  teleutospore  of  the  rusts  and  in  the  basidium,  as  Blackman 
believes,  we  have  a  synapsis  stage  followed  by  a  double  division  of  the 
nucleus  leading  to  the  formation  of  four  spores.  The  parallelism 
between  the  teleutospore  and  spore  mother  cells  of  the  higher  plants 
is  thus  complete,  and  it  is  justifiable  to  assume  that  the  first  and  second 
divisions  in  the  promycelia  and  basidia  are  respectively  heterotypic  and 
homceotypic  divisions.  On  the  other  hand,  in  the  ascus  following  the 
very  conspicuous  synapsis  described  above,  we  have  a  triple  division 
of  the  primary  nucleus  of  the  ascus  leading  to  the  formation  of  eight 
ascospore  nuclei  and  typically  to  the  formation  of  eight  spores.  The 
synapsis  stage  suggests  that  chromosome  reduction  occurs  in  the  ascus ; 
but  in  view  of  the  absolutely  universal  occurrence  of  only  two  divisions 
associated  with  chromosome  reduction  elsewhere  in  both  plant  and 
animal  kingdoms,  this  triple  division  in  the  ascus  must  be  regarded  as 
a  most  aberrant  occurrence,  and  has  led  to  very  great  hesitancy  on  my 
part  in  my  earlier  investigations  in  assuming  the  possibility  of  an  alter- 
nation of  generations  in  the  Ascomycetes  comparable  to  that  in  the 
higher  plants.  With  the  discovery  in  Phyllactinia  that  the  chromo- 
somes are  not  only  permanent  structures  of  the  cell,  but  that  they  each 
have  permanent  and  distinct  connection  with  the  central  body  through 
the  resting  condition  of  the  nuclei  as  well  as  through  the  processes  of 
both  nuclear  fusion  and  division,  combined  with  the  evidence  which  has 
been  accumulating  so  rapidly  from  all  sources  that,  even  when  appar- 
ently not  connected  with  the  centers,  the  chromosomes  still  maintain 
their  identity  in  all  nuclei,  both  of  animals  and  plants,  it  becomes  evident 
that  the  triple  division,  in  connection  with  chromosome  reduction  in  the 
ascus,  as  compared  with  a  double  division  everywhere  else  among  plants 
and  animals,  becomes  a  fact  of  still  more  fundamental  importance. 
There  is  general  agreement  at  present  that  the  chromosomes  of  the 
spore  mother  cells  are  bivalent  structures  and  that  both  heterotypic  and 


82  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

homceotypic  divisions  are  necessary  to  reduce  them  to  the  condition 
found  in  ordinary  somatic  cells.  The  three  divisions  of  the  nucleus 
of  the  ascus  following  each  other  in  rapid  succession,  just  as  do  the 
heterotypic  and  homoeotypic  divisions,  suggest  at  once  that  the  organ- 
ization of  the  chromosomes  in  this  case  must  differ  from  that  in  the 
ordinary  spore  mother  cell.  The  most  natural  assumption  would  seem 
to  be  that  the  chromosomes  are  quadrivalent  in  the  nucleus  of  the  ascus 
rather  than  bivalent,  as  in  ordinary  spore  mother  cells,  and  that  in  two 
of  the  divisions  in  the  ascus  chromosomes  are  separated  instead  of  in 
one  division,  as  in  the  ordinary  case.  Direct  evidence  as  to  whether 
the  reductions  occur  in  the  first  and  second  or  in  the  second  and  third 
divisions  is  very  difficult  to  obtain.  I  have  not  as  yet  been  able  to  recog- 
nize in  the  figures  in  the  ascus  the  ordinary  distinctions  between  hetero- 
typic and  homoeotypic  divisions. 

In  any  case,  however,  the  triple  division  certainly  suggests  that 
two  reductions  may  be  necessary  to  bring  the  chromosomes  of  each 
nucleus  back  to  the  ordinary  number,  and  this  implies  that  each  of  the 
eight  chromosomes,  as  they  emerge  from  synapsis,  represents  four 
somatic  chromosomes. 

If  from  this  standpoint  we  seek  an  explanation  of  such  a  pecul- 
iarity in  the  chromosomes  of  the  primary  nucleus  of  the  ascus,  we  are 
led  at  once  to  the  suggestion  that  they  must  have  arisen  by  a  double 
nuclear  fusion,  such  as  actually  occurs  in  the  oogonium  and  in  the  ascus, 
as  I  have  described.  We  see  thus  that  the  assumption  that  the  ascus, 
with  its  triple  division,  is  a  spore  mother  cell,  representing  the  stage  at 
which  chromosome  reduction  occurs  at  the  close  of  a  sporophytic  gen- 
eration, leads  naturally  to  the  expectation  of  two  nuclear  fusions  in  the 
development  of  the  ascocarp.  The  universality  with  which  the  triple 
division  occurs  in  all  asci  so  far  studied  is  sufficient  proof  of  its  funda- 
mental and  primitive  nature  and  that  it  occurs  in  a  spore  mother  cell 
following  an  apparent  numerical  reduction  of  the  chromosomes,  and  a 
synapsis  stage  is  in  perfect  harmony  with  the  assumption  that  it  is  neces- 
sary for  the  true  reduction  of  the  chromosome  number. 

The  triple  division  of  the  primary  nucleus  of  the  ascus  is  a  univer- 
sally present  characteristic  of  all  the  higher  Ascomycetes.  It  occurs, 
none  the  less,  whether  eight  or  a  smaller  number  of  ascospores  are  to 
be  formed,  and  that  it  is  a  process  of  fundamental  importance  is  thus 
most  strikingly  shown,  since  it  necessitates,  as  we  have  seen,  the  destruc- 
tion by  disintegration  of  six  of  the  eight  nuclei  formed  in  such  cases  as 
Phyllactinia,  in  which  but  two  spores  are  produced  in  each  ascus.  It 
is  evident  in  such  cases  that  the  three  divisions  form  together  a  single 


ALTERNATION    OF    GENERATIONS.  83 

process,  and  that  they  all  are  necessary  for  its  accomplishment.  If,  for 
example,  the  two  first  divisions  alone  were  necessary  to  accomplish  the 
reduction  of  the  chromosomes,  we  must  suppose  that  the  third  division 
would  readily  disappear  when  only  four  or  fewer  spores  were  to  be 
formed.  The  fact  that  all  three  divisions  persist  shows  their  necessity 
for  the  process  of  reduction. 

We  are  confronted  here  with  cases  parallel  to  those  in  the  higher 
plants,  where  fewer  than  four  macrospores  are  to  grow  and  become 
functional.  In  such  cases  we  find  a  strong  tendency  to  the  persist- 
ence of  double  division  as  such,  the  supernumerary  macrospores 
becoming  abortive  and  being  absorbed.  The  persistence  of  the  triple 
division  in  the  ascus  thus  suggests  in  itself  that  the  primary  nucleus 
of  the  ascus  contains  proportionally  more  chromosomes  than  the  ordi- 
nary spore  mother  cell  in  other  sporophytes,  and  we  are  thus  led  to 
expect,  what  we  actually  find,  the  two  nuclear  fusions  in  the  develop- 
ment of  the  ascocarp. 

Further,  on  analogy  with  the  spore  mother  cells  in  the  higher  plants, 
we  must  conclude  that  the  apparent  number  of  chromosomes  appearing 
in  the  divisions  immediately  succeeding  synapsis  represents  the  normal 
somatic  number,  which  in  the  case  of  Phyllactinia  would  thus  be  eight. 
I  have  above  pointed  out  the  difficulties  involved  in  counting  the  number 
of  chromosomes  in  the  ordinary  vegetative  divisions  in  the  mildews. 
The  best  one  can  say  at  present  is  that  the  appearances  are  not  against 
the  assumption  that  there  are  eight  chromosomes  on  each  half  of  the 
spindle  in  such  a  figure  as  is  shown  in  fig.  23. 

It  is  quite  certain  that  there  are  eight  or  more  chromatin  strands 
representing  chromosomes  in  the  nuclei  which  fuse  in  the  ascus.  The 
process  of  nuclear  fusion  in  the  case  of  nuclei  whose  chromosomes  are 
permanently  attached  to  the  centers,  as  I  have  found  them  in  the  mil- 
dews, leads  naturally  to  their  approximation  side  by  side  in  pairs,  and 
it  seems  probable  that  the  fusion  of  the  male  and  female  nuclei  in  the 
oogonium,  in  which  the  same  attachment  between  centers  and  chromo- 
somes exists,  would  have  the  same  result.  The  male  and  female  chro- 
mosomes would  thus  be  brought  together  in  pairs.  If,  as  in  the  case 
of  other  sexual  fusions,  the  chromosomes  brought  together  in  fertiliza- 
tion still  maintain  their  identity  till  the  stage  of  reduction  at  the  close 
of  the  sporophyte  generation,  we  must  conclude  that  the  eight  chro- 
matin strands  found  in  the  nuclei  of  the  ascus  just  before  their  fusion 
are  really  double,  and  that  in  the  fusion  in  the  ascus  these  double  chro- 
mosomes, becoming  approximated  in  pairs  and  passing  through  synap- 
sis, become  quadrivalent  chromosomes  in  the  primary  nucleus  of  the 


84  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

ascus.  These  would  then  be  in  turn  distributed  in  the  triple  division 
which  follows.  The  apparent  number  of  chromosomes  in  each  nucleus 
throughout  the  life  of  the  mildew  would  then  be  8.  The  real  number 
in  the  nuclei  in  the  ascogonium  and  ascogenous  hyphas  would  be  16, 
and  in  the  primary  nucleus  of  the  ascus  32,  as  the  size  of  these  nuclei 
indicates. 

It  is  a  matter  of  great  importance  to  determine,  in  the  nuclear 
divisions  immediately  following  fertilization,  whether,  as  would  be 
expected  by  analogy  with  the  higher  plants,  the  16  chromosomes  may 
not  be  made  out  as  distinct  units.  It  seems  probable,  however,  judging 
from  the  nature  of  the  process  as  seen  in  the  fusion  in  the  ascus,  that 
the  union  of  nuclei  with  an  organization  such  as  is  found  in  Phyllac- 
tinia  must  always  result  in  an  immediate  apparent  reduction  of  the 
chromosome  number,  though  an  actual  reduction  is  only  effected  here, 
as  in  the  higher  plants,  by  a  process  of  synapsis,  with  its  succeeding 
reduction  divisions.  These  latter  considerations,  of  course,  have  only  a 
hypothetical  value  in  the  absence  of  an  exact  determination  of  the  num- 
ber of  chromosomes  in  the  cases  involved,  and  it  is  important  that  the 
number  of  chromosomes  appearing  in  the  first  and  the  succeeding  divi- 
sions of  the  egg-nucleus  should  be  determined.  I  have  been  unable  so  far 
to  find  figures  of  this  division  which  permit  of  counting  the  number  of 
chromosomes  with  certainty.  The  division  figures  in  the  young  ascogo- 
nium seem  especially  hard  to  find  and  are  very  liable  to  show  the  chro- 
mosomes so  closely  bunched  together  that  it  is  difficult  to  make  out  their 
number. 

I  have  observed  in  a  number  of  ascogonia,  after  fertilization  in  the 
early  stages  of  their  growth,  a  small  nucleus,  one-third  or  less  the  diam- 
eter of  the  ordinary  nuclei,  whose  fate  I  have  been  unable  to  determine. 
It  is  possible  that  the  first  division  in  the  oogonium  gives  rise  to  a  super- 
numerary nucleus,  such  as  Klebahn  has  described  as  being  formed  in 
the  germination  of  the  zygospore  of  Closterium.  It  is,  of  course,  also 
of  great  importance  to  trace  out  in  detail  the  behavior  of  the  central 
bodies  here,  both  in  the  sexual  fusion  and  the  divisions  which  immedi- 
ately follow,  and  I  hope,  either  in  the  case  of  Phyllactinia  or  other 
favorable  material,  to  be  able  to  throw  further  light  on  these  questions. 
At  present  we  can  conclude,  from  the  number  of  chromosomes  in  the 
division  following  synapsis  in  the  ascus,  that  the  somatic  number  is  8, 
and,  further,  that  at  least  8  presumably  bivalent  chromosomes  are  pres- 
ent in  each  of  the  nuclei  which  fuse  in  the  ascus. 

If  we  attach  the  significance  which  I  have  indicated  above  to  the 
triple  nuclear  division  in  the  ascus,  and  if  we  further  assume  that  the 


ALTERNATION    OF    GENERATIONS.  85 

nuclear  fusion  in  the  ascus  had  at  first  no  sexual  significance,  but  arose 
in  connection  with  a  process  of  inhibited  cell  and  nuclear  division  inci- 
dent to  the  relatively  excessive  nutrition  supplied  to  the  ascus,  the  rela- 
tions of  the  young  ascus,  teleutospore,  and  basidium  become  at  once 
intelligible.  They  are  all  to  be  interpreted  morphologically  as  spore 
mother  cells.  In  the  ascus  the  triple  division  of  the  nucleus  is  necessi- 
tated by  the  fact  of  the  two  nuclear  fusions  from  which  it  has  arisen. 
But  in  the  teleutospore  and  basidium,  since  the  process  of  conjugate 
division  has  worked  back,  as  described  above,  from  its  origin  at  the 
close  of  the  sporophyte  generation  to  the  fertilization  with  which  it 
began,  and  one  nuclear  fusion  has  thus  disappeared,  we  find  the  normal 
double  division  which  occurs  elsewhere  in  spore  mother  cells.  The 
disappearance  of  a  nuclear  fusion  in  the  egg  would  thus  be  correlated 
with  the  disappearance  of  a  third  division  in  the  spore  mother  cell. 
The  conditions  in  the  rusts  and  the  conditions  in  the  mildews  afld 
Pyronema  thus  represent  two  stages  in  the  evolution  of  a  sporophyte  in 
the  higher  fungi,  of  which  that  shown  in  the  ascocarp  is  the  more  primi- 
tive. There  is,  however,  as  I  have  pointed  out  above,  no  sufficient 
evidence  in  this  to  lead  us  to  infer  that  Ascomycetes  and  Basidiomycetes 
must  be  forced  into  the  same  phylogenetic  series. 

If  it  is  true  that  the  rusts  have  arisen  from  the  red  algae  and  the 
fusion  in  the  teleutospore  is  to  be  interpreted  as  I  have  suggested,  we 
must  probably  assume,  as  noted  above,  that  in  some  of  their  ancestral 
forms  two  nuclear  fusions  are  present  in  the  life  cycle.  In  their  present 
condition  it  is  plain  that  they  are  much  changed  from  the  red  algae. 
We  have  nothing  in  the  red  algae  which  can  be  directly  compared  to 
such  series  of  carpogonia,  each  with  its  special  trichogyne  and  forming 
a  single  row  of  spores,  as  Blackman  assumes  to  represent  a  stage 
just  preceding  the  present  condition  of  the  secidium.  Blackman  has 
certainly  strengthened  the  evidence  that  the  spermatia  are  sexual  cells, 
and  that  they  once  functioned  in  fertilizing  a  trichogyne-like  organ. 
This  conclusion,  however,  leads  us  to  assume  some  still  more  primitive 
condition  than  the  sorus  of  carpogonia  which  he  conceives.  We  must 
believe  that  at  some  period  the  aecidium  was  more  like  a  cystocarp  and 
that  a  single  trichogyne  sufficed  for  the  fertilization  of  the  entire  struc- 
ture, whether  by  ooblastema  filaments,  as  in  many  of  the  red  algae 
to-day,  or  by  the  development  of  a  common  procarpic  hypha  from 
which,  when  fertilized,  the  entire  spore  mass  of  the  aecidium  arose  in  a 
fashion  analogous  to  the  method  of  origin  of  an  apothecium  of  Collema. 
The  discovery  by  Richards  (80)  of  what  is  apparently  a  carpogonial 
branch  in  the  young  aecidium  cup  of  Uromyces  caladii  and  other  forms 


86  SEXUAL    REPRODUCTION    IN    CERTAIN    MILDEWS. 

gives  us  a  possible  connecting  link  between  the  cystocarp  and  the  cseoma 
type  of  secidium.  It  may  be  that  in  the  cup-shaped  secidia  branches 
bearing  gametes  arise  from  a  true  carpogonium,  while  in  indeterminate 
aecidia  of  the  caeoma  type  this  carpogonial  branch  has  disappeared. 

If  the  evidence  advanced  by  Mottier  and  Williams  that  a  reduction 
division  occurs  in  the  development  of  the  tetrasporange  be  confirmed, 
it  is  plain  that  the  homologue  of  the  ascus  and  teleutospore,  and  of  the 
basidium  as  well,  is  in  the  tetrasporange  and  not  in  the  carpospore,  as 
many  have  been  inclined  to  assume.  The  carpospores  must  be  inter- 
preted as  conidia  intercalated  in  the  life  cycle  of  the  sporophyte,  just 
as  are  the  aecidiospores  and  uredospores  in  the  sporophyte  of  the  rust. 
In  this  case  a  close  relationship  may  be  assumed  between  the  aecidium 
and  the  cystocarp,  each  being  an  asexual  stage  appearing  early  in  the 
life  history  of  the  sporophyte  and  having  no  analogous  stage  in  the 
sporophyte  of  the  moss  or  fern.  The  ascus,  on  the  other  hand,  would 
correspond  to  the  tetrasporange,  and  there  would  be  no  stage  in  the 
Ascomycetes  to  correspond  to  the  carpospores  of  the  red  algge.  We 
should  thus  be  relieved  of  the  difficulty  of  assuming  relationship  between 
the  carpospores  produced  as  buds  or  chains  of  cells  and  the  ascospores 
formed  internally  by  free  cell  formation.  If  the  tetrasporange  is  the 
progenitor  of  the  ascus  we  must  assume  that  free  cell  formation,  with 
an  abundance  of  epiplasm,  has  been  developed  in  the  latter  for  the  dis- 
semination of  the  spores  and  as  an  adaptation  to  a  terrestrial  habit,  and 
that  the  basidium  and  teleutospore  may  have  arisen  from  the  tetraspo- 
range in  a  similar  and  parallel  series  of  developmental  forms. 

At  what  stage  in  the  evolution  of  the  Ascomycetes  from  the  red 
algae  the  nuclear  fusions  in  the  ascus  originated  is  not  apparent  from  any 
facts  yet  available.  It  may  be  found  that  some  of  the  simpler  Asco- 
mycetes, whose  asci  are  reported  as  regularly  producing  only  four  spores 
the  triple  division  has  been  replaced  by  the  ordinary  double  division  of 
spore  mother  cells.  In  that  case  it  may  be  expected  that  the  fusion  of 
nuclei  in  the  ascus  will  also  be  lacking.  It  is  quite  possible,  however, 
that  this  stage  is  not  represented  by  any  forms  at  present  known. 

The  conceptions  thus  developed  form  a  consistent  and  harmonious 
account  of  the  development  of  the  ascocarp  and  bring  the  main  facts 
as  to  its  nature  and  origin  into  line  with  what  has  been  learned  in  other 
groups  as  to  the  nature  of  the  processes  of  fertilization,  chromosome 
reduction,  the  permanence  of  the  chromosomes  and  the  central  bodies 
as  cell  structures,  and  the  nature  of  the  alternation  of  generations. 
In  these  theoretical  considerations  I  have  aimed,  of  course,  to  do  no 
more  than  endeavor  to  correlate  the  facts  as  we  know  them  to-day,  and 


ALTERNATION    OF   GENERATIONS.  87 

thus  attempt  to  systematize  our  very  fragmentary  knowledge  of  the 
higher  thallophytes ;  and  I  am  very  far  from  believing  that  such  specu- 
lations will  be  so  fortunate  as  to  achieve  a  confirmation  in  all  respects. 
Such  observations  as  Blackman's  and  Christman's  on  the  aecidium  and 
Mottier's  on  the  tetrasporange  are  sufficient  to  indicate  how  far  from 
complete  our  knowledge  is,  even  on  some  of  the  most  commonly  studied 
structures  and  processes  in  these  plants.  My  conceptions  as  to  alterna- 
tion of  generations  in  the  higher  fungi  may  be  briefly  stated  as  follows : 

In  the  rusts  we  have  sexual  reproduction  by  vegetative  fertiliza- 
tion. The  fusing  cells  are  perhaps  morphologically  vegetative  offshoots 
of  an  egg-cell.  Alternation  of  generations  is  present  and  the  sporo- 
phyte  generation  is  modified  by  the  development  of  conjugate  nuclear 
division  through  its  whole  cycle,  resulting  in  a  failure  of  the  sexual  pro- 
nuclei  to  fuse  at  the  time  of  fertilization.  Conjugate  division  may  have 
arisen  here,  as  it  is  perhaps  beginning  in  Pyronema,  immediately  prior 
to  a  nuclear  fusion  in  the  spore-mother  cell,  and  have  worked  backward 
through  the  sporophyte,  thus  tending  to  give  more  and  more  of  a  func- 
tional and  sexual  significance  to  the  fusion  in  the  spore  mother  cell, 
until,  finally  reaching  the  fertilized  egg,  the  fusion  of  the  pronuclei 
disappeared. 

In  the  Basidiomyctes  by  apogamy  sexual  cell  fusion  may  have  dis- 
appeared or  we  may  have  vegetative  fertilization.  The  sporophyte  cells 
arise  as  ordinary  hyphal  cells  which  become  binucleated,  and  conjugate 
nuclear  division  extends  through  the  entire  sporophyte  generation. 

In  the  Ascomycetes  we  have  sexual  reproduction  and  alternation 
of  generations,  modified  by  the  adaptation  of  the  spore  mother  cell  as 
an  explosive  organ  for  the  dissemination  of  the  spores  and  as  a  storage 
reservoir  for  the  production  of  resting  spores  with  a  large  supply  of 
metaplasmic  reserve  products.  The  development  of  the  relatively 
gigantic  size  of  the  ascus  led,  on  the  principle  of  the  nucleo-cytoplasmic 
relation,  to  the  increase  of  its  nuclear  content  by  inhibition  of  cell 
division  and  to  the  fusion  of  sporophyte  nuclei ;  and  this  in  turn  neces- 
sitated a  triple  instead  of  a  double  division  in  the  reduction  of  the 
chromosome  number. 

MADISON,  WISCONSIN,  April,  1905. 


INDEX  OF  LITERATURE. 


1.  ALI.EN,  C.  E.    The  early  stages  of  the  spindle  formation  in  the  pollen  mother 

cells  of  Larix.     Ann.  Bot,  xvn,  281,  1903. 

2.  Chromosome-reduction  in  Lilium  canadense.     Bot.  Gaz.,  xxxvii,  464, 

1904. 

3.  BARKER,  B.  T.  P.     Morphology  and  development  of  the  ascocarp  of  Monascus. 

Ann.  Bot.,  xvn,  167,  1903. 

4.  The  development  of  the  ascocarp  in  Ryparobius.    Report  Brit.  A.  A. 

S.     Southport,  p.  849,  1903.     (See  also  Report  for  1904.) 

5.  BAUR,  E.    Untersuchungen  iiber  die  Entwicklungsgeschichte  der  Flechten- 

apothecien.  i.    Bot.  Zeit.,  1904,  i,  21-44;  2  pi- 

6.  BENEDEN,  E.  VAN.     Recherches  sur  la  maturation  de  1'ceuf,  la  division  cellu- 

laire,  etc.    Leipzig,  1883. 

7.  BENEDEN,  E.  VAN  et  NEYT,  A.    Nouvelles  recherches  sur  la  fecondation  et  la 

division  mitosique  chez  1'Ascaride  megalocephale.    Leipzig,  1887. 

8.  BOOKMAN,  V.  H.     On  the  fertilization,  alternation  of  generations,  and  gen- 

eral cytology  of  the  Uredineae.     Ann.  Bot.,  xvm,  1904. 

9.  On  the  fertilization,  alternation  of  generations,  and  general  cytology 

of  the  Uredinese.     New  Phytologist,  in,  23,  1904. 

10.  BovERi,  TH.     Zellen  Stud.,  n.     Die  Befruchtung  u.     Teilung  des  Eies  von 

Ascaris  megalocephala.    Jena,  1888. 

11.  Zur  Physiologic  der  Kern-  und  Zellteilung.     SB.  phys.-med.  Gesell., 

Wiirzburg,  Jahrg.  1896-1897. 

12.  Uber  mehrpolige   Mitosen   als   Mittel   zur  Analyse   des   Zellkernes. 

Verh.  phys.-med.  Gesell.,  Wurzburg,  1902,  24  p. 

13.  Ergebnisse  iiber  die  Konstitution  der  chromatischen   Substanz  des 

Zellkerns.    Jena,  1904. 

14.  CHMIELEWSKI,  W.  F.     Materiaux  pour  servir  a  la  morphologic  et  physiologic 

des  proces  sexuels  chez  les  plantes  inferieurs.     1890. 

15.  CHRISTMAN,  A.  H.    Sexual  reproduction  in  the  rusts.    Bot.  Gaz.,  xix,  p.  207, 

April,  1905. 

16.  CLAUSSEN,    P.    Zur    Entwicklungsgeschichte    der    Ascomyceten.     Boudiera. 

Bot.  Zeit.,  Jahrg.,  1905,  s.  i. 

17.  CONKLIN,  E.  G.     Karyokinesis  and  cytokinesis  in  the  maturation,  fertilization, 

and  cleavage  of  Crepidula  and  other  Gasteropoda.    Jour,  of  Acad.  of 
Nat.  Sciences  of  Phila.,  2d  series,  xn,  p.  1-122,  pi.  i-vi;  1902. 

18.  DALE,  Miss  E.     Observations  on  the  Gymnoascaceas.     Ann.  Bot.,  xvn,  571, 1903. 

19.  DANGEARD,  P.  A.     La  reproduction  sexuelle  chez  les   Basidiomycetes.     Le 

Botaniste,  46  serie,  p.  87.     25  Janvier,  1895. 

20.  La  sexualite  dans  le  genre  Monascus.  ^  Le  Bot.,  96.  ser.,  p.  28,  1903. 

21.  Recherches  sur  le  developpement  du  perithece  chez  les  Ascomycetes. 

Le  Bot.,  96.  ser.,  p.  59,  1904. 

22.  DAVIS,   B.    M.    The   relationships   of   sexual   organs   in   plants.    Bot.   Gaz., 

xxxvui,  241-264,  1904. 

23.  DRUNER,  L.     Studien  uber  den  Mechanismus  der  Zellteilung.    Jen.  Zeitschr.  f. 

Naturw.,  xxix  (N.  F.  22),  p.  271-344,  1905.    Dated  1904. 

24.  FARROW,  W.  G.,  and  SEYMOUR,  A.  B.     Provisional  host  index  of  the  fungi  of 

the  United  States.     1888-1891. 

25.  FI.EMMING,  W.    Neue  Beitrage  zur  Kenntniss  der  Zelle.,  n.    Arch.  f.  Mikr. 

Anat.,  xxxvii,  685,  1891. 

26.  GERASSIMOFP,  J.  J.    Uber  den  Einfluss  des  Kerns  auf  das  Wachstum  der  Zelle. 

Bull.  Soc.  Nat.  Imp.  Mosc.     1901,  Nr.  1-2,  p.  185-220. 

27.  •    Die  Abhangigkeit  der  Grosse  der  Zelle  von  der  Menge  der  Kern- 

masse.     Zeitschr.  f.  allg.  Physiol.  i,  p.  220-258.     1902. 

88 


INDEX   OF    LITERATURE.  89 

28.  Zur  Physiologic  der  Zelle.    Bull.  Soc.  Imp.  Nat.  Mosc.    No.  I,  p.  134, 

1904. 

29.  -    —    Uber  die  Grosse  des  Zellkerns.     Beih.  zum  Bot.  Centr.,  xvm,  43,  1904. 

30.  GREGOIRE,  V.     La  reduction  numerique  des  chromosomes  et  les  cineses  de 

maturation.     La  Cellule,  xxi,  p.  297,  1904. 

31.  GUILUERMOND,  A.     Contribution  a  1'etude  cytologique  des  Ascomycetes.     C.  R. 

de  1'Acad.  des  Sci.,  vol.  137,  p.  938-939  and  1088;  1903. 

32.  •    Nouvelles   recherches    sur   1'epiplasme   des   Ascomycetes.     C.   R.    de 

TAcad.  des  Sci.,  vol.  136,  p.  1487-1489;  1903. 

33.  Sur  la  Karyokinese  de  Pezize  rutilans.    C.  R.  de  la  Soc.  de  Biol., 

LVI,  412,  1904. 

34.  Contribution  a  1'etude  de  I'epiplasme  des  Ascomycetes  et  recherches 

sur  les  corpuscles  metachromatiques  des  Champignons.    Annales  My- 
cologici,  i,  p.  201-215,  pi.  vi-vn;  1903. 

35.  HARPER,  R.  A.     Beitrage  zur  Kenntniss  der  Kernteilung  und  Sporenbildung 

im  Ascus.     Ber.  d.  Deutsch.  Bot  Gesell.,  xm,  67,  1895, 

36.  •    Die  Entwicklung  des  Peritheciums  bei  Sphaerotheca  castagnei.    Ber. 

d.  Deutsch.  Bot.  Gesell.,  xm,  475,  1895. 

37.  —     —    Uber  das  Verhalten  der  Kerne  bei   der  Fruchtentwicklung  einiger 

Ascomyceten.     JB.  f.  wiss.  Bot.,  xxix,  656,  1896. 

38.  Kernteilung  u.  freie  Zellbildung  im  Ascus.    JB.  f.  wiss  Bot.,  xxx, 

249,  1897. 

39.  Nuclear  phenomena  in  certain  stages  in  the  development  of  the  smuts. 

Trans.  Wis.  Acad.  Sci.,  xn,  475,  1899. 

40.  Sexual  reproduction  in  Pyronema  confluens  and  the  morphology  of 

the  ascocarp.     Ann.  Bot,  xiy,  p.  321-400;  pi.  19-21;  1900. 

400.  Binucleated  cells  in  certain  Hymenomycetes.     Bot.  Gaz.,  xxxm,  I, 

1902. 

41.  HEIDENHAIN,   M.     Neue  Untersuchungen  tiber  die  Centralkorper.    Arch.  f. 

mikr.  Anat,  XLIII,  423,  1894. 

42.  HENKING,  H.    Uber  Spermatogenese  u.  deren  Beziehung  zur  Entwicklung  bei 

Pyrrhocoris    apterus   L.   Zeitschr.    f.    wiss.    Zool.,   LI,  p.   685-741 ;    pi. 
35-37;  1891- 

43.  HERMANN,  F.     Beitrag  zur  Lehre  von  der  Entstehung  der  karyokinetischen 

Spindel.     Arch.  f.  mikr.  Anat.,  xxxvii,  569,  1891. 

44.  HERTWIG,  R.    Was  veranlasst  die  Befruchtung  bei  Protozoen.     SB.  Gesell. 

Morph.  u.  Phys.,  Miinchen;  xi,  p.  62-69;  1899. 

45.  Ueber  Korrelation  von  Zell-  und  Kerngrosse  und  ihre  Bedeutung 

fur  die  geschlechtliche  Differenzierung  der  Zelle.     Biol.  Cent,  xxm, 
49-62,  1903. 

46.  HOLDEN,  R.  J.,  and  HARPER,  R.  A.     Nuclear  division  and  nuclear  fusion  in 

Coleosporium    sonchi-arvensis    Lev.    Trans.    Wis.    Acad.    Sci.,    xrv, 
63,  1903- 

47.  IKENO,  S.    Die  Sporenbildung  von  Taphrina-Arten.    Flora  oder  Allg.     Bot. 

Zeit,  xcn,  p.  1-31;  pl.  i-3;  1903- 

48.  Uber  die  Sporenbildung  und  systematische  Stellung  von  Monascus 

purpureus  Went.    Ber.  d.  Deutsch.  Bot.  Gesell.,  xxi,  259,  1903. 

49.  JANSSENS.   F.   A.    La   Spermatogenese  chez  les   Tritons.    La  Cellule,   xix, 

fasc.  i,  pp.  5-116,  1902. 

50.  JENKINSON,  J.  W.    Observations  on  the  maturation  and  fertilization  of  the 

egg  of  the  Axolotl.    Quart.  Jour.  Mic.  Sci.,  XLVIII,  428,  1904. 

51.  JuEL,  H.  O.     Taphridium  Lagerh.  und  Juel.     Eine  neue  Gattung  der  Proto- 

mycetaceen.     Bihang  till  K.  Svenska  Vet  Akad.  Handlmger,  xxvn, 
i   1902 

52.  'Uber   Zellinhalt,    Befruchtung   und    Sporenbildung   bei    Dipodascus. 

Flora.    Erganzungsbd.    p.  47;  J9O2. 

53.  KOSTANECKI,  K.   V.,  and  WIERZYSKY,  A.    Uber  das  Verhalten  der  sogen. 

achromatischen   Substanzen  im  befruchteten  Ei  nach  Beobachtungen 
an  Physa  fontinalis.     Arch.  f.  mikr.  Anat.,  XLVII,  309,  1896. 

54.  KOSTANECKI,  K.  V.    Uber  die  Bedeutung  der  Polstrahlen  wahrend  der  Mitose. 

Arch.  f.  mikr.  anat,  XLIX,  p.  651-706;  pl.  19-20;  1897 


9O  INDEX   OF    LITERATURE. 

55.  KUYPER,   H.    P.     Die    Perithecium   Entwickelung   von    Monascus   purpureus 

Went  und   Monascus   Barker!  Dang,   und  die  systematische   Stellung 
dieser   Pilze.    Ext.   du  Recueil   des  Travaux  botanique   Neerlandais, 

1904,  2-4. 

56.  De   perithecium — ontwikkeling   van    Monascus   purpureus    Went   en 

Mon.  Barkeri  Dang.     Kon.  Akad.  von  Weten.,  Amsterdam,  xm,  46, 
May  28,  1904. 

57.  LINDAU.    Nat.  Wochenschr.,  Nr.  27,  April  3,  1904,  p.  425. 

58.  LUBOSCH.     Uber  die  Eireifung  d.  Metazoen.     Ergeb.  der  Anat.  und  Entwick., 

Merkel  u.  Bonnet,  n,  709,  1901. 

59.  MAIRE,   R.     Recherches   cytologiques    sur   le   Galactinia   succosa.     C.    R.    de 

1'Acad  des  Sci.  Paris,  vol.  137  p.  769-771 ;  1903. 

60.  La  formation  des  asques  chez  les   Pezizes  et  1'evolution  nucleaire 

des  Ascomycetes.     C.  R.  de  1.  Soc.  de  Biologic,  LV,  p.  1401,  Nov.,  1903. 

61.  Sur  les  divisiones  nucleaires  dans  1'Asque  de  la  Morille  et  de  quelques 

autres  Ascomycetes.     C.  R.  de  1.  Soc.  d.  Biologic,  LVI,  p.  822,   1904. 

62.  Recherches   cytologiques   et   taxonomiques    sur   les    Basidiomycetes. 

Lons-le-Saunier,  1902. 

63.  MEVES,  F.    Uber  die  Entwicklung  der  mannlichen  Geschlechtszellen  von  Sala- 

mandra  maculosa.    Arch.  f.  mikr.  Anat.,  XLVIII,  i,  1897. 

64.  MoLLER,  A.     Phycomyceten  u.  Ascomyceten.     Jena,  1901. 

65.  MONTGOMERY,  T.  H.    The  spermatogenesis  in  Pentatoma  up  to  the  formation 

of  the  spermatid.    Zool.,  JB.,  xn,  Abt.  f.  Anat,  p.  1-88;  pi.  1-5;  1898. 

66.  •    A  study  of  the  chromosomes  of  the  germ  cells  of  Metazoa.    Trans. 

Am.  Phil.  Soc..,  xx,  p.  154-236;  pi.  4-8;  1901. 

67.  MOTTIER,  D.  M.    tNuclear  and  cell  division  in  Dictyota  dichotoma.     Ann.  Bot., 

xiv,  103,  1900. 

68.  NEGER,  F.  W.     Beitrage  zur  Biologic  der  Erysipheen.     Flora,  LXXXVIII,  333, 

1901 ;  and  xc,  221,  1902. 

69.  Neue  Beobachtungen  iiber  das  spontane  Freiwerden  der  Erysipheen- 

fruchtkorper.     Centr.   f.   Bakt.   Parasit.   u.   Infekt,   1903;   2.    Abt   x, 
570-573- 

70.  NEMEC,  B.    Ueber  die  Einwirkung  des  Chloralhydrates  auf  die  Kern-  und 

Zellteilung.    JB.  fur  wiss.  Bot,  xxxix,  645,  1904. 

71.  NICHOLS,  S.  P.    The  nature  and  origin  of  the  binucleated  cells  in  certain 

Basidiomycetes.    Trans.  Wis.  Acad.  Sci.,  xv,  1905. 

72.  OLIVE,  E.  W.     The  morphology  of  Monascus  purpureus.     Bot  Gaz.,  xxxix, 

56,  1005. 

73.  OvERTON,  E.    Uber  die  qsmotischen  Eigenschaften  der  Zelle  in  ihrer  Bedeu- 

tung  fur  die  Toxikologie  und  Pharmakologie.     Festschr.  Nat  Gesell. 
Zurich,  1896. 

74.  Uber  die  allgemeinen  osmotischen  Eigenschaften  der  Zelle,  ihre  ver- 

mutlichen  Ursache  u.   ihre  Bedeutung  fur  die   Physiologic.     Viertel- 
jahrschr.  Nat.  Gesell.  Zurich,  Jahrg.,  XLIV,  88,  1899. 

75.  PAULMIER,  F.  C.    The  spermatogenesis  of  Anasa  tristis.    Journ.  Morph.,  xv, 

1899,  p.  223-269;  pi.  13-14. 

76.  PALLA,  E.     Uber  die   Gattung  Phyllactinia.   Ber.   d.   Deutsch.   Bot.   Gesell., 

xvn,  64-72,  1899. 

77.  POIRAULT,  G.  and  RACIBORSKI,  M.     Sur  les  noyaux  des  Uredinees.     Extr. 

Journ.  de  Botanique,  96  annee,  pp.  1-21 ;  1895. 

78.  RABL,  C.    Uber  Zellteilung.     Morphologisches  Jahrbuch,  x,  1885. 

79.  •    Uber  Zellteilung  Anat.  Anz.,  rv,  21,  1889. 

80.  RICHARDS,  H.  M.     On  some  points  in  the  development  of  the  Aecidia.     Proc. 

Am.  Acad.  Arts  and  Sci.,  xxxi,  255,  1896. 

81.  ROSENBERG,  O.    Ueber  die  Individuality  der  Chromosomen  im  Pflanzenreich. 

Flora  oder  Allg.  Bot.  Zeitung,  xciu,  251,  1904. 
810.  Zur    Kenntniss    der   Reduktions-Teilung   in    Pflanzen.      Bot.    Not, 

1905,  p.  1-24. 

82.  SALMON,  E.  S.    A  monograph  of  the  Erysiphaceae.    Mem.  Torr.  Bot.  Club, 

ix,  1900. 

83.  Supplementary  notes   on   the  Erysiphese.     Bull.   Torrey   Bot.   Club, 

xxix,  181. 


INDEX   OF    LITERATURE.  9! 

84.  SAPPIN-TROUFFY.    Recherches   histologiques   sur   la   famille   des   Uredinees. 

Le  Bot,  5e  ser.,  1896,  pp.  59-244. 

85.  SCHAPER,  A.     Beitrage  zur  Analyse  des  tierischen  Wachstums  I.  Theil.  Arch. 

f.  Entwick.-Mech.,  Bd.,  xiv,  pp.  307-400,  pi.  15-25;  1902. 

86.  SMITH,  GRANT.    The  Haustoria  of  the  Erysipheae.     Bot.  Gaz.,  xxix,  153,  1900. 

87.  STRASBURGER,  E.    Zellbildung  u.  Zellteilung,  3  Aufl.,  pp.  26-27.    Jena,  1880. 

Uber  Kern-  und  Zelltheilung  im  Pflanzenreich.     Hist.  Beitr.,  I,  Jena, 


89.  Uber  die  Wirkungssphare  der  Kerne  u.  die  Zellgrosse.     Hist.  Beitr. 

v,  pp.  95-124;  Jena,  1893. 

90.  •    Uber   Reduktionsteilung,    Spindelbildung,    Centrosomen   und   Cilien- 

bildner  im  Pflanzenreich.     Hist.  Beitr.,  vi,  Jena,  1900. 

91.  .    Ueber   Reduktionstheilung.     SB.   d.   Kon.    Preuss.   Acad.   d.   Wiss., 

1904,  I.  Halbbd.  pp.  587-614.  , 

92.  SUTTON,  M.  S.    On  the  morphology  of  the  chromosome  group  in  Brachystola 

magna.     Biol.  Bull.,  iv,  pp.  24-39;  1902. 

920.  SWINGLE,  W.  T.    Zur  Kenntniss  der  Kern-  und  Zellteilung  bei  den  Sphace- 
lariaceen.    JB.  f.  wiss.  Bot,  xxx,  297,  1897. 

93.  THAXTER,   R.     Contributions  toward   a  monograph  of  the   Laboulbeniaceae. 

Mem.  Am.  Acad.  Arts  and  Sci.,  xn,  187. 

94.  TIEGHEM,  Ph.  van.    L'ceuf  des  plantes  considere  comme  base  de  leur  class- 

ification.   Ann.  de  Sci.  Nat.  Bot.,  8.  ser.,  xiv,  pp.  213-390;  1901. 

95.  TROW,   A.   H.    On    fertilization    in    the    Saprolegnieae.    Ann.    Bot,    xvm, 

541,  1904. 

96.  VRIES,  H.  de.     Befruc'htung  u.  Bastardierung.      Liepzig,  1903. 

97.  WAGER,  H.    The  sexuality  of  the  Fungi.    Ann.  Bot,  xm,  pp.  29-55,  1899. 

98.  WILLIAMS,  J.  L.     Studies  in  the  Dictyotaceae.    Ann.  Bot,  xvni,  141,  1904. 

99.  WOLFE,  J.  J.     Cytological  studies  on  Nemalion.    Ann.  Bot.,  xvin,  607,  1904. 

100.  WOYCICKI,  Z.     Einige  neue  Beitrage  zur   Entwicklungsgeschichte  von  Basi- 

diobolus  ranarum.     Flora,  xcm,  87,  1904. 


92  EXPLANATION    OF    FIGURES    IN    PLATES. 

[All  figures  were  drawn  with  the  Abbe  camera  lucida  and,  with  the  exception  of 
fig.  30,  with  the  Zeiss  apochromatic  objective,  2  mm.  n.  ap.  1.40;  figs,  i,  3-11,  25, 
44-47,  55-6i,  65-78  with  compens.  oc.  8;  figs.  2, 12-20, 22, 51  with  oc.  o ;  figs.  21,  26-29 
with  oc.  4;  figs.  23,  24,  50,  52-54,  59,  62-64,  79  with  oc.  12;  figs.  31-42,  48,  49  with 
oc.  18;  fig.  30  obj.  8  mm.  oc.  8.] 

PI,ATE  I. — PHYLLACTINIA  CORYLEA. 

FIG.  i. — Male  and  female  branches ;  the  gametes  not  yet  cut  off  by  septa. 

FIG.  2. — Same,  showing  hyphal  cell,  from  which  the  oogonium  arises,  most  of 
the  oogonial  branch  cut  away. 

FIG.  3. — The  oogonial  branch  coiled  around  the  antheridial  branch;  the  latter 
septate  at  the  level  at  which  it  is  constricted  by  the  oogonial  branch. 

FIG.  4.— The  nucleus  of  the  male  branch  has  divided  to  form  the  antheridial 
and  stalk-cell  nuclei. 

FIG.  5. — The  antheridium  has  been  cut  off  from  the  stalk-cell  and  lies  above 
and  to  one  side  of  the  apex  of  the  oogonium. 

FIG.  6. — 'Slightly  older  than  stage  of  fig.  5.  Antheridium  and  stalk  partly  behind 
the  oogonium. 

FIG.  7. — Antheridium  and  oogonium  just  before  conjugation,  the  former  behind 
the  apex  of  the  latter. 

FIG.  8. — Stage  of  conjugation.  The  section  lies  in  the  plane  of  contact  of  the 
antheridium  and  the  apex  of  the  oogonium.  The  conjugation-pore  is  present  and 
appears  as  a  circular  opening  through  the  walls  of  the  gametes.  The  male  nucleus 
is  in  contact  with  the  larger  egg  nucleus. 

FIG.  9. — Stage  of  conjugation  a  little  later  than  fig.  8.  The  section  cuts  the 
plane  of  contact  of  the  gametes  at  right  angles.  The  conjugation-pore  is  open, 
and  through  it  the  protoplasts  of  the  gametes  form  a  continuous  mass  of  cyto- 
plasm; a  large  vacuole  in  the  antheridium  just  outside  the  conjugation-pore.  The 
pronuclei  are  in  contact;  the  male  nucleus  larger  than  in  fig.  8,  but  still  smaller 
than  the  egg-nucleus. 

FIG.  10. — Oogonium  with  single  large  fusion  nucleus.  Cytoplasm  of  anther- 
idium still  spongy  but  containing  some  granules. 

FIG.  ii. — Oogonium  still  uninucleated.    Wall  of  antheridium  beginning  to  swell. 

FIG.  12. — Oogonium  still  uninucleated.  Wall  of  antheridium  swollen  and  show- 
ing strong  affinity  for  orange  stain.  Early  stage  of  perithecial  walls. 

FIG.  13. — Egg-nucleus  has  divided. 

FIG.  14. — Ascogonium  binucleated.  Stalk-cell  of  antheridium  has  grown  out 
into  a  hyphal  branch,  which  curves  over  the  antheridium  and  is  a  part  of  the 
perithecial  wall. 

FIG.  15  a,  b. — Two  sections  of  the  young  ascocarp.  Antheridium  with  thick  wall 
and  dense  content 

FIG.  16. — Slightly  older ;  perithecial  wall  becoming  two-layered. 

FIG.  17  a,  b. — Young  ascocarp  in  two  sections ;  uninucleated  cell  cut  off  from 
the  end  of  multinucleated  ascogonium;  perithecial  hypha  is  shown  arising  from 
stalk  of  antheridium. 

FIG.  180, b. — Somewhat  older;  end  cell  of  ascogonium  separated  from  the 
antheridium  by  crowding  in  of  the  perithecial  hyphae. 

FIG.  19.— Ascogonium  with  three  cells,  the  next  to  the  last  binucleated,  the 
others  uninucleated.  The  whole  ascocarp  is  perhaps  dwarfed  in  this  case. 


PLATE 


94  EXPLANATION    OF    FIGURES    IN    PLATES. 


PLATE  II. — PHYLLACTINIA  CORYLEA. 

FIG  20  a,  b. — Ascogonium  with  four  cells ;  the  apical  cell  uninucleated  and  nar- 
rowed above  by  pressure  of  surrounding  hyphae.  Penultimate  cell  binucleated, 
nucleus  of  basal  cell  in  next  section. 

FIG.  21  a,  b. — Ascogonium  four-celled ;  basal  cell  with  two  nuclei. 

FIG.  22. — Ascogonium  with  five  cells ;  penultimate  cell  budding  out  in  asco- 
genous  hyphae ;  nuclei  of  second  and  third  cells  in  another  section. 

FIG.  23. — Perithecial  cells  with  resting  nuclei  and  a  metaphase  stage  of  nuclear 
division ;  nucleolus  near  one  pole  of  spindle. 

FIG.  24. — Resting  nucleus  from  mycelial  hypha  with  central  body  connected  with 
chromatin. 

FIG.  25  a,  b. — Ascogonium  with  five  cells ;  penultimate  cell  budding  out  in  asco- 
genous  hyphae;  apical  cell  still  connected  to  thick-walled  antheridium. 

FIG.  26. — Median  section  of  older  ascocarp,  showing  sections  of  ascogenous 
hyphae,  antheridium,  etc. ;  peripheral  cells  swollen,  in  some  cases  in  preparation 
for  pushing  out  as  hyphal  branches. 

FIG.  27. — Median  section  of  still  older  ascocarp,  showing  portion  of  ascogonium 
and  sections  of  multinucleated  ascogenous  hyphae. 

FIG.  28. — Section  showing  cells  of  ascogonium  and  ascogenous  hyphae  at  stage 
when  latter  become  septate. 

FIG.  290,  b. — Sections  showing  the  pushing  out  of  the  cells  of  the  ascogenous 
hyphae  to  form  the  young  asci. 

FIG.  30. — Median  section  of  ascocarp  just  after  fusion  of  nuclei  in  the  young 
asci;  two  or  more  layers  of  cells  around  the  asci,  with  dense  content  and  thin 
walls ;  peripheral  cells  on  upper  surface  of  ascocarp  swelling  out  to  form  penicil- 
late  cells. 


HARPER. 


$6  EXPLANATION    OF    FIGURES    IN    PLATES. 


PLATE   III. — PHYLLACTINIA  CORYLEA. 

FIG.  31. — Young  ascus  with  two  nuclei;  central  bodies  facing  each  other. 

FIG.  32. — Slightly  older  ascus ;  chromatin  strands  more  conspicuous. 

FIG.  33. — Just  before  nuclear  fusion  in  ascus.  Lower  nucleus  lies  above  and 
partially  overlapping  the4  other. 

FIG.  34. — iSection  of  ascus  with  slice  of  one  nucleus  showing  three  chromatin 
strands  and  sections  of  others. 

FIG.  35. — Early  stage  in  fusion  of  nuclei;  central  body  of  upper  nucleus  has 
pushed  ahead  on  the  membrane  of  the  lower  nucleus,  its  chromatin  system  drawn 
out  in  long  cone. 

FIG.  36. — Stage  of  fusion.  Central  bodies  and  chromatin  of  two  nuclei  side  by 
side,  but  independent;  nucleoli  already  combined  in  one. 

FIG.  37. — Chromatin  systems  overlapping,  but  distinct.     Nucleoli  in  contact. 

FIG.  38. — (Stage  just  before  the  central  bodies  combine;  nucleoli  in  this  case 
still  separate. 

FIG.  39.— The  central  bodies  are  joined  side  by  side.  Chromatin  systems  com- 
bining. 

FIG.  40. — Central  bodies  and  chromatin  systems  completely  combined.  Nucleoli 
still  separate. 

FIG.  41. — Shows  entire  ascus ;  stage  about  the  same  as  in  fig.  40. 

FIG.  42.-^Fusion  of  nuclei  complete. 


98  EXPLANATION    OF    FIGURES    IN    PLATES. 


PLATE  IV. — PHYLLACTINIA  CORYLEA. 

FIG.  43. — Early  stage  in  synapsis. 

FIG.  44. — 'Synapsis ;  ends  of  chromatin  strands  protruding  from  contracted  mass. 
FIG.  45. — Late  synapsis  stage  stained  to  specially  differentiate  the  central  body. 
FIG.  46. — Loosening  up  of  chromatin  after  synapsis. 
FIG.  47. — Early  spirem  stage. 

FIG.  48. — Culmination  of  spirem  stage;  eight  distinct  chromatin  strands  corre- 
sponding to  eight  chromosomes  of  later  stages. 

FIG.  49. — Same  stage;  strands  spreading  from  center  without  forming  cone. 
FIG.  50. — Young  penicillate  cell.    Three  nuclei  showing  central  bodies. 
FIG.  51. — Full-grown  penicillate  cell. 

FIG.  52. — Transverse  section  of  ascus,  showing  prophase  in  formation  of  spindle. 
FIG.  53. — Primary  nucleus  of  ascus ;  equatorial-plate  stage,  eight  chromosomes. 


HARPER. 


PLATE  IV. 


\ 
\ 

\ 


R.  A.  H.  del. 


IOO  EXPLANATION    OF    FIGURES    IN    PLATES. 


PLATE  V. — PHYU,ACTINIA  CORYLEA. 

FIG.  54. — Primary  nucleus  of  ascus,  early  metaphase. 

FIG.  55. — Anaphase,  eight  chromosomes  on  each  half  of  the  spindle.  Polar 
aster  strongly  developed. 

FIG.  56. — lyate  anaphase. 

FIG.  57. — Diaster;  Chromosomes  still  attached  to  pole  by  spindle  fibers. 

FIG.  58. — Reconstruction  of  daughter  nuclei;  chromosomes  elongating  and 
becoming  irregular  strands  of  chromatin. 

FIG.  59. — Later  stage;  strands  of  chromatin  forming  irregular  cone  extending 
from  central  body  into  antipolar  region  of  nucleus. 


HARPER. 


1O2  EXPLANATION    OF    FIGURES    IN    PLATES. 


PLATE   VI. — PHYLLACTINIA  CORYLEA. 

FIG.  60. — Chromatin  strands  more  irregular  and  passing  over  into  apparent 
reticulum  by  formation  of  anastomoses. 

FIG.  61. — Still  later,  chromatin  still  more  irregular  and  practically  in  the  resting 
condition. 

FIG.  62. — Transverse  section  of  ascus ;  nucleus  in  spirem  of  second  division. 

FIG.  63  a,  b. — Later  prophase  of  second  division ;  chromosomes  appearing  in  two 
sections. 

FIG.  64. — 'Still  later  prophase,  second  division. 

FIG.  65. — Equatorial-plate  stage;  second  division;  one  spindle  lying  in  plane  of 
section,  the  other  cut  through  obliquely ;  eight  chromosomes. 

FIG.  66. — Late  anaphase  stages,  second  division;  eight  chromosomes  on  each 
half  of  each  spindle. 

FIG.  67  a. — Equatorial-plate  stages,  third  division;  nuclei  in  end  of  ascus;  one 
polar  and  one  side  view  of  the  spindle  figures. 

FIG.  67  b. — Larger  nuclear  figure  from  same  ascus,  which  will  form  functional 
nuclei ;  nuclear  membrane  still  intact. 

FIG.  68. — Anaphase  stages  of  third  division ;  eight  chromosomes  on  each  spindle- 
half. 


HARPER. 


K.  A.  H.  del. 


IO4       EXPLANATION  OF  FIGURES  IN  PLATES. 


PLATE    VII— PHYUvACTINIA  CORYLEA,    EXCEPT    WHERE    OTHERWISE    INDICATED. 

FIG.  69. — Erysiphe  cichoracearum.  Dispirem  stages;  third  division;  polar  asters 
strongly  developed. 

FIG.  70. — Beaked  nucleus  and  aster  in  early  stage  of  spore  formation ;  two  super- 
numerary nuclei  at  opposite  end  of  ascus  and  lying  with  their  central  bodies  on 
the  plasma-membrane  of  the  ascus. 

FIG.  71. — Beaked  nucleus  and  aster,  opposite  which  the  plasma-membrane  is 
drawn  in  forming  an  oval  depression. 

FIG.  72. — Beaked  nucleus  with  aster  strongly  developed;  central  body  broader 
than  end  of  beak. 

FIG.  73. — Erysiphe  cichoracearum.  Metamorphosis  of  the  polar  aster  to  form 
the  plasma-membrane  of  the  spore;  early  stage  in  folding  over  of  the  rays;  two 
supernumerary  nuclei  present;  transverse  section  of  part  of  membrane  of  third 
spore  shown  in  lower  part  of  figure. 

FIG.  74. — E.  cichoracearum;  the  rays  have  formed  broad,  bell-shaped  membranes 
about  the  nuclei ;  beak  of  nucleus  lying  to  the  right  is  cut  off  and  appears  in  the 
next  section. 

FIG.  75. — E.  cichoracearum;  plasma-membrane  of  spore  almost  complete. 

FIG.  76. — Spore  completely  inclosed;  beak  of  nucleus  still  attached  to  plasma- 
membrane  ;  astral  rays  have  disappeared  entirely. 

FIG.  77. — 'Stage  in  drawing  in  of  nuclear  beak;  central  body  conspicuous  at  the 
tip  of  beak. 

FIG.  78. — Later  stage,  same  process. 

FIG.  79. — Ascus  with  two  spores;  nucleus  in  resting  condition;  chromatin  con- 
spicuously oriented  on  central  body. 

FIG.  80. — E.  communis;  late  stage  in  spore  formation;  central  body  and  system 
of  astral  rays  divided;  beak  scarcely  visible. 

FIG.  81. — E.  communis;  ascus  with  view  of  stage  in  spore  formation  looking 
somewhat  obliquely  down  upon  the  end  of  the  spore;  two  supernumerary  nuclei 
in  opposite  end  of  ascus. 


HARPER. 


U.C.  BERKELEY  LIBRARIES 


MJ246562 


THE  UNIVERSITY  OF  CALIFORNIA  LIBRARY 


